Treatment And Inhibition Of Inflammatory Lung Diseases In Patients Having Risk Alleles In The Genes Encoding IL33 And IL1RL1

ABSTRACT

IL33 antagonists alone or in combination with IL-4R antagonists can be used to treat or inhibit eosinophilic asthma, eosinophilic COPD, eosinophilic ACOS, and nasal polyps in a subject having one or more risk alleles in the intronic IL1RL1 variant rs1420101, in the IL33 variant rs1342326, in both, or in variants in linkage disequilibrium thereof.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing filed electronically as anXML file named 381203758SEQ, created on Jun. 7, 2023, with a size of423,000 bytes. The Sequence Listing is incorporated herein by reference.

FIELD

This disclosure relates generally to the field of precision medicine.More particularly, the disclosure relates to the detection of riskalleles in genes encoding IL33 and IL1RL1, which risk alleles can beused to stratify inflammatory lung disease patients as having a highrisk of developing one or more of these conditions and theireosinophilic subtypes.

BACKGROUND

Various publications, including patents, published applications,accession numbers, technical articles and scholarly articles are citedthroughout the specification. Each of these cited publications isincorporated by reference, in its entirety and for all purposes, in thisdocument.

Asthma and Chronic Obstructive Pulmonary Disease (COPD) are highlyprevalent obstructive lung diseases with substantial unmet clinical needand significant diagnostic overlap, and there is increasing interest inthe intersection of these conditions, termed asthma-COPD overlapsyndrome (ACOS). There is a long-standing debate as to whether the twodiseases have a shared etiology (the so-called “Dutch Hypothesis”) orhave independent mechanistic causes (the so-called “British Hypothesis).Despite recent progress in elucidating genetic contribution to commoncomplex disease risk, including obstructive lung diseases, there is nowell-established genetic finding linking asthma to COPD.

Genome-wide association studies (GWAS) have identified common geneticvariants at interleukin-33 (IL33) and/or IL1RL1 that are associated withasthma. IL33, a pro-inflammatory cytokine and member of theinterleukin-1 (IL-1) cytokine family, is expressed in subsets of cellsin barrier tissues, including the lung epithelium. IL33 signals via aheterodimeric receptor complex composed of the IL33-specific receptorIL1RL1 (also known as ST2 or IL33R) and the IL-1RAcP co-receptor, commonto several receptors of the IL-1 family.

In damaged tissues, previously sequestered IL33 is passively releasedinto the extracellular compartment by necrotic cells and functions as anendogenous “danger signal” (alarmin) that activates inflammatory andrepair pathways. Cigarette smoke induces IL33 expression in lungepithelial cells in mice, and IL33 expression is elevated in thebronchial epithelium of both asthma and COPD patients. In disease statesin which inflammatory infiltrate and inflammatory cytokines are alreadypresent, the pool of IL33-responsive cells is increased and IL33signaling further amplifies immune responses, resulting in pathologicinflammation and exaggerated immune responses, potentially drivingchronic inflammatory diseases such as COPD.

There is also an asthma-COPD overlap syndrome (ACOS), characterized bysymptoms common to both asthma and COPD. Nevertheless, clinicalchallenges remain in the capacity to diagnose ACOS, given the difficultyin separating asthma from COPD owing to the overlapping features incommon.

Treatment challenges for asthma, COPD, and ACOS also remain, withresistance to corticosteroids (the standard of care) fairly commonplace.As well, other treatments such as IL-5 therapy has not worked well forthe eosinophilic subsets of asthma and COPD.

Accordingly, there remains a need in the art to distinguish amongasthma, COPD, and ACOS, as well as to more accurately identify patientswho have the eosinophilic subsets of these disorders. Proper diagnosescan better direct a therapeutic regimen and improve patient outcomes.

SUMMARY

In a first aspect of the disclosure, a method for treating or inhibitingeosinophilic asthma comprises administering an IL33 antagonist oradministering an IL33 antagonist and an IL-4R antagonist to a subjecthaving one or more risk alleles associated with eosinophilic asthma inthe intronic IL1RL1 variant rs1420101 (SEQ ID NO: 357) or variant inlinkage disequilibrium thereof, in the IL33 variant rs1342326 (SEQ IDNO: 358) or variant in linkage disequilibrium thereof, or in both theintronic IL1RL1 variant rs1420101 (SEQ ID NO: 357) or variant in linkagedisequilibrium thereof and the IL33 variant rs1342326 (SEQ ID NO: 358)or variant in linkage disequilibrium thereof. Administration of an IL33antagonist and/or an IL-4R antagonist is such that eosinophilic asthmais treated or inhibited in the subject.

In a second aspect of the disclosure, a method for treating orinhibiting eosinophilic Chronic Obstructive Pulmonary Disease (COPD)comprises administering an IL33 antagonist or administering an IL33antagonist and an IL-4R antagonist to a subject having one or more riskalleles associated with eosinophilic COPD in the intronic IL1RL1 variantrs1420101 (SEQ ID NO: 357) or variant in linkage disequilibrium thereof,in the IL33 variant rs1342326 (SEQ ID NO: 358) or variant in linkagedisequilibrium thereof, or in both the intronic IL1RL1 variant rs1420101(SEQ ID NO: 357) or variant in linkage disequilibrium thereof and theIL33 variant rs1342326 (SEQ ID NO: 358) or variant in linkagedisequilibrium thereof. Administration of an IL33 antagonist and/or anIL-4R antagonist is such that eosinophilic COPD is treated or inhibitedin the subject.

In a third aspect of the disclosure, a method for treating or inhibitingeosinophilic asthma-Chronic Obstructive Pulmonary Disease (COPD) overlapsyndrome (ACOS), comprising administering an IL33 antagonist oradministering an IL33 antagonist and an IL-4R antagonist to a subjecthaving one or more risk alleles associated with eosinophilic asthma inthe intronic IL1RL1 variant rs1420101 (SEQ ID NO: 357) or variant inlinkage disequilibrium thereof, in the IL33 variant rs1342326 (SEQ IDNO: 358) or variant in linkage disequilibrium thereof, or in both theintronic IL1RL1 variant rs1420101 (SEQ ID NO: 357) or variant in linkagedisequilibrium thereof and the IL33 variant rs1342326 (SEQ ID NO: 358)or variant in linkage disequilibrium thereof. Administration of an IL33antagonist and/or an IL-4R antagonist is such that eosinophilic COPD istreated or inhibited in the subject.

In a fourth aspect of the disclosure, a method for treating orinhibiting nasal polyps comprises administering an IL33 antagonist oradministering an IL33 antagonist and an IL-4R antagonist to a subjecthaving one or more risk alleles associated with nasal polyps in theintronic IL1RL1 variant rs1420101 (SEQ ID NO: 357) or variant in linkagedisequilibrium thereof, in the IL33 variant rs1342326 (SEQ ID NO: 358)or variant in linkage disequilibrium thereof, or in both the intronicIL1RL1 variant rs1420101 (SEQ ID NO: 357) or variant in linkagedisequilibrium thereof and the IL33 variant rs1342326 (SEQ ID NO: 358)or variant in linkage disequilibrium thereof. Administration of an IL33antagonist and/or an IL-4R antagonist is such that nasal polyps aretreated or inhibited in the subject.

In a fifth aspect of the disclosure, a method for assessing risk ofdevelopment of eosinophilic asthma, eosinophilic Chronic ObstructivePulmonary Disease (COPD), or eosinophilic asthma COPD overlap syndrome(ACOS) comprises the steps of:

(A) detecting one or more risk alleles associated with eosinophilicasthma, eosinophilic COPD, or eosinophilic ACOS in the intronic IL1RL1variant rs1420101 (SEQ ID NO: 357) or variant in linkage disequilibriumthereof, in the IL33 variant rs1342326 (SEQ ID NO: 358) or variant inlinkage disequilibrium thereof, or in both the intronic IL1RL1 variantrs1420101 (SEQ ID NO: 357) or variant in linkage disequilibrium thereofand the IL33 variant rs1342326 (SEQ ID NO: 358) or variant in linkagedisequilibrium thereof in a sample obtained from a subject;

(B) (i) assigning a risk score of 1 to the subject when the subject hasa risk allele in the intronic IL1RL1 variant rs1420101 (SEQ ID NO: 357)or variant in linkage disequilibrium thereof in one of the chromosome 2homologs or a risk allele in the IL33 variant rs1342326 (SEQ ID NO: 358)or variant in linkage disequilibrium thereof in one of the chromosome 9homologs,

(B) (ii) assigning a risk score of 2 to the subject when the subject hasa risk allele in the intronic IL1RL1 variant rs1420101 (SEQ ID NO: 357)or variant in linkage disequilibrium thereof in both of the chromosome 2homologs, when the subject has a risk allele in the IL33 variantrs1342326 (SEQ ID NO: 358) or variant in linkage disequilibrium thereofin both of the chromosome 9 homologs, or when the subject has a riskallele in the intronic IL1RL1 variant rs1420101 (SEQ ID NO: 357) orvariant in linkage disequilibrium thereof in one of the chromosome 2homologs and a risk allele in the IL33 variant rs1342326 (SEQ ID NO:358) or variant in linkage disequilibrium thereof in one of thechromosome 9 homologs,

(B) (iii) assigning a risk score of 3 to the subject when the subjecthas a risk allele in the intronic IL1RL1 variant rs1420101 (SEQ ID NO:357) or variant in linkage disequilibrium thereof in both of thechromosome 2 homologs and a risk allele in the IL33 variant rs1342326(SEQ ID NO: 358) or variant in linkage disequilibrium thereof in one ofthe chromosome 9 homologs, or when the subject has a risk allele in theintronic IL1RL1 variant rs1420101 (SEQ ID NO: 357) or variant in linkagedisequilibrium thereof in one of the chromosome 2 homologs and a riskallele in the IL33 variant rs1342326 (SEQ ID NO: 358) or variant inlinkage disequilibrium thereof in both of the chromosome 9 homologs, or

(B) (iv) assigning a risk score of 4 to the subject when the subject hasa risk allele in the intronic IL1RL1 variant rs1420101 (SEQ ID NO: 357)or variant in linkage disequilibrium thereof in both of the chromosome 2homologs and a risk allele in the IL33 variant rs1342326 (SEQ ID NO:358) or variant in linkage disequilibrium thereof in both of thechromosome 9 homologs; and

(C) categorizing the subject's risk of development of eosinophilicasthma, eosinophilic COPD, or eosinophilic ACOS, wherein a risk score of1 indicates that the subject has a risk of developing thehigh-eosinophilic subset of eosinophilic asthma, high-eosinophilicsubset of eosinophilic COPD, or high-eosinophilic subset of eosinophilicACOS, a risk score of 2 indicates that the subject has an elevated riskof developing the high-eosinophilic subset of eosinophilic asthma,high-eosinophilic subset of eosinophilic COPD, or high-eosinophilicsubset of eosinophilic ACOS, a risk score of 3 indicates that thesubject has a high risk of developing the high-eosinophilic subset ofeosinophilic asthma, high-eosinophilic subset of eosinophilic COPD, orhigh-eosinophilic subset of eosinophilic ACOS, and a risk score of 4indicates that the subject has a very high risk of developing thehigh-eosinophilic subset of eosinophilic asthma, high- eosinophilicsubset of eosinophilic COPD, or high-eosinophilic subset of eosinophilicACOS. The method may further comprise treating or inhibiting one or moreof the eosinophilic asthma, eosinophilic COPD, or eosinophilic ACOS,including the high eosinophilic subset thereof, in the subject byadministering to the subject an IL33 antagonist or an IL33 antagonistand an IL-4R antagonist.

In a sixth aspect of the disclosure, an IL33 antagonist or a combinationof an IL33 antagonist and an IL-4R antagonist is for use in thetreatment or inhibition of, or in the manufacture of a medicament forthe treatment or inhibition of any one of eosinophilic asthma,eosinophilic Chronic Obstructive Pulmonary Disease (COPD), eosinophilicasthma-Chronic Obstructive Pulmonary Disease overlap syndrome (ACOS),high-eosinophil eosinophilic asthma, high-eosinophil eosinophilic COPD,high-eosinophil eosinophilic ACOS, or nasal polyps when a patientthereof has one or more risk alleles associated with eosinophilicasthma, eosinophilic COPD, or eosinophilic ACOS in the intronic IL1RL1variant rs1420101 (SEQ ID NO: 357) or variant in linkage disequilibriumthereof, in the IL33 variant rs1342326 (SEQ ID NO: 358) or variant inlinkage disequilibrium thereof, or in both the intronic IL1RL1 variantrs1420101 (SEQ ID NO: 357) or variant in linkage disequilibrium thereofand the IL33 variant rs1342326 (SEQ ID NO: 358) or variant in linkagedisequilibrium thereof.

According to any one of these aspects, the subject may have at least onerisk allele associated with eosinophilic asthma in the intronic IL1RL1variant rs1420101 (SEQ ID NO: 357) or variant in linkage disequilibriumthereof, may have two risk alleles associated with eosinophilic asthmain the intronic IL1RL1 variant rs1420101 (SEQ ID NO: 357) or variant inlinkage disequilibrium thereof, may have at least one risk alleleassociated with eosinophilic asthma in the IL33 variant rs1342326 (SEQID NO: 358) or variant in linkage disequilibrium thereof, or may havetwo risk alleles associated with eosinophilic asthma in the IL33 variantrs1342326 (SEQ ID NO: 358) or variant in linkage disequilibrium thereof,and may have further at least one risk allele associated witheosinophilic asthma in the IL33 variant rs1342326 (SEQ ID NO: 358) orvariant in linkage disequilibrium thereof, and/or may further have tworisk alleles associated with eosinophilic asthma in the IL33 variantrs1342326 (SEQ ID NO: 358) or variant in linkage disequilibrium thereof.

According to any one of these aspects, the method may comprise or theuse may be for administering an IL33 antagonist to the subject or themethod may comprise or the use may be for administering an IL33antagonist and an IL-4R antagonist to the subject. The IL33 antagonistmay comprise an IL33 trap or an antibody that specifically binds toIL33. The IL-4R antagonist may comprise an antibody that specificallybinds to IL-4R.

According to any of these aspects, the IL33 trap may comprise a firstIL33 binding domain comprising an IL33 binding portion of IL1RL1 and asecond IL33 binding domain comprising an extracellular portion ofIL-1RAcP. According to any of these aspects, the antibody orantigen-binding fragment thereof that specifically binds to IL33 maycomprise the H1, H2, and H3 domains of SEQ ID NO: 274 and the L1, L2,and L3 domains of SEQ ID NO: 282. According to any of these aspects, theantibody or antigen-binding fragment thereof that specifically binds toIL-4R may comprise the H1, H2, and H3 domains of SEQ ID NO: 337 and theL1, L2, and L3 domains of SEQ ID NO: 338.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (Panels A, B, C, and D) shows four exemplary arrangements of theindividual components of the IL33 antagonists relative to one another.Panel A shows an arrangement in which a first IL33-binding domain (D1)is attached to the N-terminus of a first multimerizing domain (M1), anda second IL33-binding domain (D2) is attached to the N-terminus of asecond multimerizing domain (M2). D1 is shown as a white box and D2 isshown as a black box to indicate that D1 and D2 are derived fromdifferent IL33 binding proteins. Panel B shows an arrangement in which afirst IL33-binding domain (D1) is attached to the N-terminus of a firstmultimerizing domain (M1), and a second IL33-binding domain (D2) isattached to the C-terminus of a second multimerizing domain (M2). D1 isshown as a white box and D2 is shown as a black box to indicate that D1and D2 are derived from different IL33 binding proteins. Panels C and Dshow arrangements comprising four IL33-binding domains, D1, D2, D3 andD4. In these arrangements, D3-D1-M1 and D4-D2-M2 are attached in tandem,wherein D3 is attached to the N-terminus of D1, and D1 is attached tothe N-terminus of M1; and D4 is attached to the N-terminus of D2, and D2is attached to the N-terminus of M2. In Panel C, D3 and D4 are identicalor substantially identical to one another, and D1 and D2 are identicalor substantially identical to one another. In Panel D, D1 and D4 areidentical or substantially identical to one another, and D3 and D2 areidentical or substantially identical to one another.

FIG. 2 (Panels A, B, C, and D) shows rs1420101 (IL1RL1, also known asST2), s1342326 (IL33) and rs146597587 (IL33-pLoF) associations with(Panel A) eosinophil counts (Panel B) login eosinophil counts. Theassociations between the total burden of rs1420101 and rs1342326 riskalleles, and (Panel C) eosinophil counts (Panel D) log₁₀ eosinophilcounts is also shown. Effect sizes and P-values for eosinophil countsand log₁₀ eosinophil counts, were calculated using linear regression,with adjustment for age, age², sex, smoking status and principalcomponents of ancestry. P-values and effect sizes/odds ratios wereestimated for individual scores; in each case the comparison was toindividuals with zero risk alleles. Additionally, overall alleliceffects p-values are shown.

FIG. 3 (Panels A, B, and C) shows rs1420101 (IL1RL1, also known as ST2),s1342326 (IL33) and rs146597587 (IL33-pLoF) associations with (Panel A)Asthma, (Panel B) High Eosinophil Asthma Subset and (Panel C) LowEosinophil Asthma Subset. Odds ratios for disease were calculated usinglogistic regression, with adjustment for age, age², sex, smoking statusand principal components of ancestry.

FIG. 4 (Panels A, B, and C) shows rs1420101 (IL1RL1, also known as ST2),s1342326 (IL33) and rs146597587 (IL33-pLoF) associations with (Panel A)COPD, (Panel B) High Eosinophil COPD Subset and (Panel C) Low EosinophilCOPD Subset. Odds ratios for disease were calculated using logisticregression, with adjustment for age, age², sex, smoking status andprincipal components of ancestry.

FIG. 5 (Panels A, B, and C) shows rs1420101 (IL1RL1, also known as ST2),s1342326 (IL33) and rs146597587 (IL33-pLoF) associations with (Panel A)ACOS, (Panel B) High Eosinophil ACOS Subset and (Panel C) Low EosinophilACOS Subset. Odds ratios for disease were calculated using logisticregression, with adjustment for age, age², sex, smoking status andprincipal components of ancestry.

FIG. 6 (Panels A, B, and C) shows genetic score (total burden ofrs1420101 and rs1342326 risk alleles) associations with (Panel A) Asthma(Panel B) COPD and (Panel C) ACOS. P-values odds ratios were estimatedfor individual scores; in each case the comparison was to individualswith zero risk alleles. Additionally, overall trend test p-values areshown.

FIG. 7 (Panels A, B, and C) shows genetic score (total burden ofrs1420101 and rs1342326 risk alleles) associations with (Panel A) Highand Low Eosinophil Asthma (Panel B) High and Low Eosinophil COPD and(Panel C) High and Low Eosinophil ACOS. P-values odds ratios wereestimated for individual scores; in each case the comparison was toindividuals with zero risk alleles. Additionally, overall trend testp-values are shown.

FIG. 8 (Panels A, B, C, and D) shows the clinical characteristics ofstudy participants stratified by capture reagent (Panel A) VCRome and(Panel B) xGEN, and chip platform (Panel C) OMNI and (Panel D) GSA.

FIG. 9 (Panels A and B) shows rs1420101 (IL1RL1, also known as ST2),s1342326 (IL33), rs146597587 (IL33-pLoF) and genetic score (total burdenof rs1420101 and rs1342326 risk alleles) associations with (Panel A)Allergic Rhinitis and (Panel B) Nasal Polyps. Odds ratios for diseasewere calculated using logistic regression, with adjustment for age,age², sex, smoking status and principal components of ancestry. To testthe burden of common risk variants, p-values and odds ratios wereestimated for each individual score; in each case the comparison was toindividuals with zero risk alleles. Additionally, overall trend testp-values are shown.

DETAILED DESCRIPTION

Various terms relating to aspects of disclosure are used throughout thespecification and claims. Such terms are to be given their ordinarymeaning in the art, unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinition provided herein.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless expressly stated otherwise.

The terms “subject” and “patient” are used interchangeably and includeany animal. Mammals are preferred, including companion (e.g., cat, dog)and farm mammals (e.g., pig, horse, cow), as well as rodents, includingmice, rabbits, and rats, guinea pigs, and other rodents. Non-humanprimates are more preferred, and human beings are highly preferred.

The term “isolated” means removed and/or altered from the naturalenvironment by the hand of a human being.

A “risk allele” includes alternative polymorphisms at a particularposition that associate with a risk of developing a disease, disorder,or condition.

“Linkage disequilibrium” refers to a nonrandom association of alleles attwo or more loci.

It has been observed in accordance with the disclosure that singlenucleotide polymorphisms in IL1R1rs1420101 (SEQ ID NO: 357) and IL33rs1342326 (SEQ ID NO: 358) associate with increased risk of asthma, aswell as high-eosinophil subsets of asthma, COPD, and ACOS. In addition,it was observed that individuals carrying a larger burden of these riskalleles across both loci have an attendant larger disease risk and,heterozygous carriers of rare pLOF variants in IL33 had lower medianlifetime eosinophil counts and trends reflecting decreased risk ofasthma, as well as trends reflecting decreased risks high-eosinophilsubsets of asthma, COPD, and ACOS. It is believed that IL33 pathwaygenetic variants have not been previously associated with COPD and, itis further believed that there have not been any reported genetic linksbetween asthma and COPD or genetic links between the IL33 pathway therisk of high-eosinophil subsets of asthma, COPD and ACOS. Furthermore,it was observed that single nucleotide polymorphisms in IL1R1rs1420101and IL33 rs1342326, considered individually and in aggregate, associatewith increased risk of nasal polyps and allergic rhinitis. These dataindicate a role for interleukin-33 blockade in the treatment ofhigh-eosinophil forms of obstructive lung diseases such as asthma, COPD,and ACOS, as well as other upper airways diseases such as nasal polypsand their high-eosinophil subsets. Accordingly, the disclosure featuresmethods for identifying risk, diagnosing, treating, and inhibitingasthma, COPD, and ACOS, especially the high-eosinophil subsets thereof.

In a first aspect, the disclosure features methods for assessing risk ofdevelopment of an inflammatory lung disease. The inflammatory lungdisease may be one or more of asthma, COPD, ACOS, or nasal polyps. Theasthma may be eosinophilic asthma or high-eosinophil eosinophilicasthma. The COPD may be eosinophilic COPD or high-eosinophileosinophilic COPD. The ACOS may be eosinophilic ACOS or high-eosinophileosinophilic ACOS. In general, the methods comprise detecting one ormore risk alleles associated with risk of development of suchinflammatory lung diseases.

In some embodiments, the methods comprise detecting one or more riskalleles associated with eosinophilic asthma, eosinophilic COPD,eosinophilic ACOS, or nasal polyps in the intronic IL1RL1 variantrs1420101 (SEQ ID NO: 357) or variant in linkage disequilibrium thereof,in the IL33 variant rs1342326 (SEQ ID NO: 358) or variant in linkagedisequilibrium thereof, or in both the intronic IL1RL1 variant rs1420101(SEQ ID NO: 357) or variant in linkage disequilibrium thereof and theIL33 variant rs1342326 (SEQ ID NO: 358) or variant in linkagedisequilibrium thereof in a sample obtained from a subject, thenassigning a risk score of 1 to the subject when the subject has a riskallele in the intronic IL1RL1 variant rs1420101 (SEQ ID NO: 357) orvariant in linkage disequilibrium thereof in one of the chromosome 2homologs or a risk allele in the IL33 variant rs1342326 (SEQ ID NO: 358)or variant in linkage disequilibrium thereof in one of the chromosome 9homologs, assigning a risk score of 2 to the subject when the subjecthas a risk allele in the intronic IL1RL1 variant rs1420101 (SEQ ID NO:357) or variant in linkage disequilibrium thereof in both of thechromosome 2 homologs, when the subject has a risk allele in the IL33variant rs1342326 (SEQ ID NO: 358) or variant in linkage disequilibriumthereof in both of the chromosome 9 homologs, or when the subject has arisk allele in the intronic IL1RL1 variant rs1420101 (SEQ ID NO: 357) orvariant in linkage disequilibrium thereof in one of the chromosome 2homologs and a risk allele in the IL33 variant rs1342326 (SEQ ID NO:358) or variant in linkage disequilibrium thereof in one of thechromosome 9 homologs, assigning a risk score of 3 to the subject whenthe subject has a risk allele in the intronic IL1RL1 variant rs1420101(SEQ ID NO: 357) or variant in linkage disequilibrium thereof in both ofthe chromosome 2 homologs and a risk allele in the IL33 variantrs1342326 (SEQ ID NO: 358) or variant in linkage disequilibrium thereofin one of the chromosome 9 homologs, or when the subject has a riskallele in the intronic IL1RL1 variant rs1420101 (SEQ ID NO: 357) orvariant in linkage disequilibrium thereof in one of the chromosome 2homologs and a risk allele in the IL33 variant rs1342326 (SEQ ID NO:358) or variant in linkage disequilibrium thereof in both of thechromosome 9 homologs, or assigning a risk score of 4 to the subjectwhen the subject has a risk allele in the intronic IL1RL1 variantrs1420101 (SEQ ID NO: 357) or variant in linkage disequilibrium thereofin both of the chromosome 2 homologs and a risk allele in the IL33variant rs1342326 (SEQ ID NO: 358) or variant in linkage disequilibriumthereof in both of the chromosome 9 homologs.

In some embodiments, the risk allele in the IL33 variant rs1342326comprises a single nucleotide polymorphism (SNP). In some detailedembodiments, the IL33 variant comprises the SNP 9:6190076:A:C. (HumanGenome GRCh38) The variant rs1342326 comprises the following nucleicacid sequence: CCAATCTTTTCTCATGAAGACACCA[G/T]CATGACCTCTTATTCTTA TTTATAT(SEQ ID NO: 358).

In some embodiments, the risk allele in the IL1RL1 variant rs1420101comprises an SNP. In some detailed embodiments, the IL1RL1 variantcomprises the SNP 2:102341256:C:T (Human Genome GRCh38). The variantrs1342326 comprises the following nucleic acid sequence:TATACCATCACAAAGCCTCTCATTA[A/G]ACTTTGAATCCAATGAGTATTACTA (SEQ ID NO:357).

Detection may be according to any suitable methodology. The risk allelesmay be detected, for example, by way of sequencing, genotyping,imputation, probing with complementary nucleic acid probes.

The methods may further comprise categorizing the subject's risk ofdevelopment of eosinophilic asthma, eosinophilic COPD, or eosinophilicACOS, wherein a risk score of 1 indicates that the subject has a risk ofdeveloping the high-eosinophil subset of eosinophilic asthma,high-eosinophil subset of eosinophilic COPD, or high-eosinophil subsetof eosinophilic ACOS, a risk score of 2 indicates that the subject hasan elevated risk of developing the high-eosinophil subset ofeosinophilic asthma, high-eosinophil subset of eosinophilic COPD, orhigh-eosinophil subset of eosinophilic ACOS, a risk score of 3 indicatesthat the subject has a high risk of developing the high-eosinophilsubset of eosinophilic asthma, high-eosinophil subset of eosinophilicCOPD, or high-eosinophil subset of eosinophilic ACOS, and a risk scoreof 4 indicates that the subject has a very high risk of developing thehigh-eosinophil subset of eosinophilic asthma, high-eosinophil subset ofeosinophilic COPD, or high-eosinophil subset of eosinophilic ACOS. Inthis scale, an elevated risk is greater than a risk but lesser than ahigh risk, and a very high risk is greater than a high risk. Thus, interms of patient risk of developing disease, risk<elevated risk<highrisk<very high risk, or risk score of 1<risk score of 2<risk score of3<risk score of 4.

The methods may further comprise obtaining a sample from the subject. Ingeneral, the sample may comprise any sample from which the risk allelesmay be detected. The sample may comprise a tissue sample or sputum. Atissue sample may include peripheral blood, airway or lung tissue.

The methods may further comprise identifying the subject as a candidatefor treatment with an IL33 antagonist or a combination of an IL33antagonist and an IL-4R antagonist. Based on the categorization of thesubject's risk as a risk score of 1, risk score of 2, risk score of 3,or risk score of 4, the subject may benefit from a therapeutic regimenthat inhibits eosinophilic asthma, eosinophilic COPD, eosinophilic ACOS,or the high eosinophil subsets thereof, or nasal polyps. An inhibitorytherapeutic regimen may comprise adjustments in type, dose, dosingfrequency, etc. for the IL33 antagonist, as well as whether or not tocombine with an IL-4R antagonist and, if so, the type, dose, and dosingfrequency, etc. for the IL-4R antagonist, for example, depending on thelevel of risk.

The methods may further comprise detecting increased levels ineosinophil counts from blood or sputum isolated from the subject.Increased levels are those that are considered above normal levels orabove levels typically observed in subjects that have thenon-eosinophilic subset of asthma, COPD, or ACOS. The methods mayfurther comprise isolating the blood or sputum from the subject for thispurpose.

The methods may further comprise administering an IL33 antagonist oradministering an IL33 antagonist and an IL-4R antagonist to the subject.Such administration may be according to an amount effective to inhibiteosinophilic asthma, eosinophilic COPD, or eosinophilic ACOS, or thehigh-eosinophil subset thereof. In some embodiments, the IL33 antagonistmay comprise an IL33 trap. In some embodiments, the IL33 antagonist maycomprise an antibody that specifically binds to IL33 or antigen-bindingfragment thereof. Suitable IL33 antagonists are described herein. Insome embodiments, the IL-4R antagonist may comprise an antibody thatspecifically binds to IL-4R or antigen-binding fragment thereof.Suitable IL-4R antagonists are described herein.

In a second aspect, the disclosure features methods for treating orinhibiting eosinophilic asthma in a subject in need thereof. Theeosinophilic asthma may be the low-eosinophil subset of eosinophilicasthma or may be the high eosinophil subset of eosinophilic asthma.

In some embodiments, the methods comprise administering an IL33antagonist or administering an IL33 antagonist and an IL-4R antagonistto a subject having one or more risk alleles associated witheosinophilic asthma in the intronic IL1RL1 variant rs1420101 (SEQ ID NO:357) or variant in linkage disequilibrium thereof, in the IL33 variantrs1342326 (SEQ ID NO: 358) or variant in linkage disequilibrium thereof,or in both the intronic IL1RL1 variant rs1420101 (SEQ ID NO: 357) orvariant in linkage disequilibrium thereof and the IL33 variant rs1342326(SEQ ID NO: 358) or variant in linkage disequilibrium thereof, such thateosinophilic asthma is treated or inhibited in the subject.

In some embodiments, the IL33 antagonist may comprise an IL33 trap. Insome embodiments, the IL33 antagonist may comprise an antibody thatspecifically binds to IL33 or antigen-binding fragment thereof. SuitableIL33 antagonists are described herein. In some embodiments, the IL-4Rantagonist may comprise an antibody that specifically binds to IL-4R orantigen-binding fragment thereof. Suitable IL-4R antagonists aredescribed herein.

In a third aspect, the disclosure features methods for treating orinhibiting eosinophilic COPD in a subject in need thereof. Theeosinophilic COPD may be the low-eosinophil subset of eosinophilic COPDor may be the high eosinophil subset of eosinophilic COPD.

In some embodiments, the methods comprise administering an IL33antagonist or administering an IL33 antagonist and an IL-4R antagonistto a subject having one or more risk alleles associated witheosinophilic COPD in the intronic IL1RL1 variant rs1420101 (SEQ ID NO:357) or variant in linkage disequilibrium thereof, in the IL33 variantrs1342326 (SEQ ID NO: 358) or variant in linkage disequilibrium thereof,or in both the intronic IL1RL1 variant rs1420101 (SEQ ID NO: 357) orvariant in linkage disequilibrium thereof and the IL33 variant rs1342326(SEQ ID NO: 358) or variant in linkage disequilibrium thereof, such thateosinophilic COPD is treated or inhibited in the subject.

In some embodiments, the IL33 antagonist may comprise an IL33 trap. Insome embodiments, the IL33 antagonist may comprise an antibody thatspecifically binds to IL33 or antigen-binding fragment thereof. SuitableIL33 antagonists are described herein. In some embodiments, the IL-4Rantagonist may comprise an antibody that specifically binds to IL-4R orantigen-binding fragment thereof. Suitable IL-4R antagonists aredescribed herein.

In a fourth aspect, the disclosure features methods for treating orinhibiting eosinophilic ACOS in a subject in need thereof. Theeosinophilic ACOS may be the low-eosinophil subset of eosinophilic ACOSor may be the high eosinophil subset of eosinophilic ACOS.

In some embodiments, the methods comprise administering an IL33antagonist or administering an IL33 antagonist and an IL-4R antagonistto a subject having one or more risk alleles associated witheosinophilic ACOS in the intronic IL1RL1 variant rs1420101 (SEQ ID NO:357) or variant in linkage disequilibrium thereof, in the IL33 variantrs1342326 (SEQ ID NO: 358) or variant in linkage disequilibrium thereof,or in both the intronic IL1RL1 variant rs1420101 (SEQ ID NO: 357) orvariant in linkage disequilibrium thereof and the IL33 variant rs1342326(SEQ ID NO: 358) or variant in linkage disequilibrium thereof, such thateosinophilic ACOS is treated or inhibited in the subject.

In some embodiments, the IL33 antagonist may comprise an IL33 trap. Insome embodiments, the IL33 antagonist may comprise an antibody thatspecifically binds to IL33 or antigen-binding fragment thereof. SuitableIL33 antagonists are described herein. In some embodiments, the IL-4Rantagonist may comprise an antibody that specifically binds to IL-4R orantigen-binding fragment thereof. Suitable IL-4R antagonists aredescribed herein.

In any of the methods described or exemplified herein, an IL33antagonist may be administered as part of a therapeutic regimen. An IL33antagonist may comprise any agent that inhibits the interaction of IL33with one or more of its binding partners and, in so doing, inhibitIL33-mediated signaling. For example, an IL33 antagonist may bind toand/or interact with IL33, or with the IL33 receptor referred to as“suppression of tumorigenicity” (aka ST2), or with the IL33 co-receptorInterleukin-1 Receptor Accessory Protein (IL-1RAcP), or with a complexof any of the following: IL33/ST2, or ST2/IL-1RAcP.

Non-limiting examples of categories of IL33 antagonists include smallmolecule IL33 inhibitors, or receptor antagonists, or nucleic acids thathybridize under stringent conditions to nucleic acid sequences encodingeither IL33, or an IL33 receptor or co-receptor (e.g., short interferingRNAs (siRNA) or clustered regularly interspaced short palindromic repeatRNAs (CRISPR-RNA or crRNA), including single guide RNAs (sgRNAs) havinga crRNA and tracrRNA sequence. Other IL33 antagonists include proteinscomprising a ligand-binding portion of an IL33 receptor (e.g., ST2),IL33-binding scaffold molecules (e.g., DARPins, HEAT repeat proteins,ARM repeat proteins, tetratricopeptide repeat proteins,fibronectin-based scaffold constructs, and other scaffolds based onnaturally occurring repeat proteins, and anti-IL33 aptamers or portionsthereof.

In preferred embodiments, an IL33 antagonist comprises an antibody thatspecifically binds to human IL33 (IL33 antibodies), or antigen-bindingfragments thereof. The amino acid sequence identifiers for exemplaryanti-IL33 antibodies for use in the methods described herein are shownin Table 1. Anti-IL33 antibodies may comprise any antibody described inU.S. Pat. No. 9,453,072, which is incorporated by reference in itsentirety.

TABLE 1 IL33 Antibodies Amino Acid Sequence Identifiers Antibody SEQ IDNOs: Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1M9559N2 4 6 8 10 12 14 16 H1M9566N 18 20 22 24 26 28 30 32 H1M9568N 34 36 3840 42 44 46 48 H4H9629P 50 52 54 56 58 60 62 64 H4H9633P 66 68 70 72 7476 78 80 H4H9640P 82 84 86 88 90 92 94 96 H4H9659P 98 100 102 104 106108 110 112 H4H9660P 114 116 118 120 122 124 126 128 H4H9662P 130 132134 136 138 140 142 144 H4H9663P 146 148 150 152 154 156 158 160H4H9664P 162 164 166 168 170 172 174 176 H4H9665P 178 180 182 184 186188 190 192 H4H9666P 194 196 198 200 202 204 206 208 H4H9667P 210 212214 216 218 220 222 224 H4H9670P 226 228 230 232 234 236 238 240H4H9671P 242 244 246 248 250 252 254 256 H4H9672P 258 260 262 264 266268 270 272 H4H9675P 274 276 278 280 282 284 286 288 H4H9676P 290 292294 296 298 300 302 304 H1M9565N 308 310 312 314 316 318 320 322

In some embodiments, the IL33 antagonist comprises an anti-IL33antibody, or antigen-binding fragment thereof, comprising a heavy chainvariable region (HCVR), light chain variable region (LCVR), and/orcomplementarity determining regions (CDRs) of the amino acid sequencesof the anti-IL33 antibodies as set forth in U.S. Pat. No. 9,453,072 andin Table 1 herein. In some embodiments, the IL33 antagonist comprisesthe heavy chain complementarity determining regions (CDR; e.g., H1, H2,H3) of the heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 274 and the light chain CDRs (e.g., L1, L2, L3)of the light chain variable region comprising the amino acid sequence ofSEQ ID NO: 282. In some embodiments, the H1 comprises the amino acidsequence of SEQ ID NO: 276, the H2 comprises the amino acid sequence ofSEQ ID NO: 278, and the H3 comprises the amino acid sequence of SEQ IDNO: 280. In some embodiments, the L1 comprises the amino acid sequenceof SEQ ID NO: 284, the L2 comprises the amino acid sequence of SEQ IDNO: 286, and the L3 comprises the amino acid sequence of SEQ ID NO: 288.In yet other embodiments, the anti-IL33 antibody or antigen- bindingfragment thereof comprises an HCVR comprising SEQ ID NO: 274 and an LCVRcomprising SEQ ID NO: 282.

In some embodiments, the IL33 antibodies or antigen-binding fragmentsthereof comprise three heavy chain CDRs (HCDR1, HCDR2 and HCDR3)contained within a heavy chain variable region (HCVR) amino acidsequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50,66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290,and 308; and comprises three light chain CDRs (LCDR1, LCDR2 and LCDR3)contained within a light chain variable region (LCVR) amino acidsequence selected from the group consisting of SEQ ID NOs: 10, 26, 42,58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282,298, and 316.

In some embodiments, the anti-IL33 antibodies, or antigen-bindingfragments thereof comprise a HCVR and LCVR (HCVR/LCVR) sequence pair ofSEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122,130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250,258/266, 274/282, 290/298, or 308/316.

In some embodiments, the anti-IL33 antibodies, or antigen-bindingfragments thereof comprise a heavy chain CDR1 (HCDR1) domain having anamino acid sequence selected from the group consisting of SEQ ID NO: 4,20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244,260, 276, 292, and 310, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; a heavy chain CDR2 (HCDR2) domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54,70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294,and 312, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity; alight chain CDR1 (LCDR1) domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108,124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, and 318, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a light chainCDR2 (LCDR2) domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142,158, 174, 190, 206, 222, 238, 254, 270, 286, 302, and 320, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity.

In some preferred embodiments, the anti-IL33 antibodies orantigen-binding fragments thereof compriseHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, having theamino acid sequences selected from the group consisting of: SEQ ID NOs:4-6-8-12-14-16 (e.g. H1M9559N); 20-22-24-28-30-32 (e.g., H1M9566N);36-38-40-44-46-48 (e.g., H1M9568N); 52-54-56-60-62-64 (e.g. H4H9629P);68-70-72-76-78-80 (e.g., H4H9633P); 84-86-88-92-94-96 (e.g. H4H9640P);100-102-104-108-110-112 (e.g., H4H9659P); 116-118-120-124-126-128 (e.g.,H4H9660P); 132-134-136-140-142-144 (e.g., H4H9662P);148-150-152-156-158-160 (e.g., H4H9663P); 164-166-168-172-174-176 (e.g.,H4H9664P); 180-182-184-188-190-192 (e.g., H4H9665P);196-198-200-204-206-208 (e.g., H4H9666P); 212-214-216-220-222-224 (e.g.,H4H9667P); 228-230-232-236-238-240 (e.g., H4H9670P);244-246-248-252-254-256 (e.g., H4H9671P); 260-262-264-268-270-272 (e.g.,H4H9672P); 276-278-280-284-286-288 (e.g., H4H9675P);292-294-296-300-302-304 (e.g., H4H9676P); and 310-312-314-318-320-322(H1M9565N).

In some embodiments, the anti-IL33 antibodies, or antigen-bindingfragments thereof, comprise the heavy and light chain CDR domainscontained within heavy and light chain variable region (HCVR/LCVR)sequences of SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106,114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234,242/250, 258/266, 274/282, 290/298, or308/316. The boundaries of CDRsmay be according to the Kabat definition, the Chothia definition, or theAbM definition.

Other anti-IL33 antibodies and antigen-binding fragments thereof thatmay be used in the methods described herein are disclosed in EuropeanPubl. No. EP 1725261, PCT Publ. Nos. WO 2011/031600, WO 2015/099175, WO2015/106080 (ANB020), WO 2016/077381, WO 2016/077366, or WO2016/156440,U.S. Pat. No. 8,187,596, and U.S. Publ. No. 2016/0168242, which are eachincorporated herein by reference in their entirety.

In alternative preferred embodiments, an IL33 antagonist comprises anIL33 trap. IL33 traps comprise at least one IL33 binding domain, whichcomprises an IL33 binding portion of an IL33 receptor protein,designated ST2. In some embodiments, an IL33 trap further comprises anextracellular portion of an IL33 co-receptor, designated IL-1 receptoraccessory protein, or IL-1RAcP. The IL33 trap may also comprise at leastone multimerizing component, which functions to connect the variouscomponents of the trap with one another. The various components of theIL33 traps are described below and shown in FIG. 1 . IL33 traps maycomprise any trap described in U.S. Publ. No. 2014/0271642 and PCT Publ.No. WO 2014/152195, which are each incorporated herein by reference intheir entirety.

The IL33 trap may comprise a first IL33 binding domain (D1) attached toa multimerizing domain (M). In some embodiments, the IL33 trap comprisesa second IL33 binding domain (D2) attached to D1 and/or M. In somepreferred embodiments, D1 comprises an IL33-binding portion of an ST2protein. In some preferred embodiments, D2 comprises an extracellularportion of an IL-1RAcP protein.

The individual components of the IL33 traps may be arranged relative toone another in a variety of ways that result in functional antagonistmolecules capable of binding IL33. For example, D1 and/or D2 may beattached to the N-terminus of M. In some embodiments, D1 and/or D2 isattached to the C-terminus of M. In other embodiments, D1 is attached tothe N-terminus of D2, and D2 is attached to the N-terminus of M,resulting in an in-line fusion, from N- to C-terminus, of an antagonistmolecule represented by the formula D1-D2-M. Other orientations of theindividual components are disclosed elsewhere herein in FIG. 1 .

The IL33 traps comprise at least one IL33 binding domain (sometimesreferred to herein by the designation “D,” or “D1,” “D2,” etc.). In someembodiments, the IL33 binding domain comprises an IL33-binding portionof an ST2 protein. An IL33-binding portion of an ST2 protein cancomprise or consist of all or part of the extracellular domain of an ST2protein. In preferred embodiments, an ST2 protein is a human ST2protein, including the ST2 protein of amino acids 1-556 of accessionnumber NP_057316.3 (SEQ ID NO: 352). In some alternative embodiments,the ST2 protein comprises an ST2 protein from a non-human species (e.g.,mouse ST2, non-human primate ST2, etc.). An preferred IL33-bindingportion of an ST2 protein is set forth herein as the amino acid sequenceof SEQ ID NO: 328 (corresponding to the extracellular domain of humanST2 [K19-S328 of NCBI Accession No. NP_057316.3]). Other examples of anIL33-binding portion of an ST2 protein is set forth herein as the aminoacid sequence of SEQ ID NO: 329 (corresponding to the extracellulardomain of mouse ST2 [S27-R332 of NCBI Accession No. P14719]).

In some embodiments, the IL33 binding domain of the trap comprises anextracellular portion of an IL-1RAcP protein. In certain embodiments, anIL-1RAcP protein comprises a human IL-1RAcP protein, including anIL-1RAcP protein having the amino acid sequence of SEQ ID NO: 353. Insome alternative embodiments, the IL-1RAcP protein comprises an IL-1RAcPprotein from a non-human species (e.g., mouse IL-1RAcP, non-humanprimate IL-1RAcP, etc.). An exemplary extracellular portion of anIL-1RAcP protein is set forth herein as the amino acid sequence of SEQID NO: 330 (corresponding to the extracellular domain of human IL-1RAcP[S21-E359 of NCBI Accession No. Q9NPH3]). Another example of anextracellular portion of an IL-1RAcP protein is set forth herein as theamino acid sequence of SEQ ID NO: 331 (corresponding to theextracellular domain of mouse IL-1RAcP [S21-E359 of NCBI Accession No.Q61730]).

Non-limiting examples of IL33 traps for use in the methods are shown inTable 2, and include the IL33 traps designated “hST2-hFc,” “hST2-mFc,”“hST2-hIL1RAcP-mFc,” “hST2-hIL1RAcP-hFc” and “mST2-mIL1RAcP-mFc.” Thesecorrespond to SEQ ID NOs: 323, 324, 325, 326 and 327, respectively. IL33receptor based traps may comprise an amino acid sequence that is atleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to any of the exemplary IL33 receptor based traps set forthherein (e.g., SEQ ID NOs: 323, 324, 325, 326 and 327). IL33 traps maycomprise D1 and/or D2 components having an amino acid sequence that isat least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to any of the exemplary IL33 binding domain componentamino acid sequences set forth herein (e.g., SEQ ID NOs: 328, 329, 330and 331).

Five different exemplary IL33 traps were constructed. The first IL33antagonist (hST2-hFc, SEQ ID NO: 323) includes the soluble extracellularregion of human ST2 (SEQ ID NO: 328) fused at its C-terminus to theN-terminus of a human IgG1 Fc region (SEQ ID NO:332). The second IL33antagonist (hST2-mFc, SEQ ID NO:324) consists of the solubleextracellular region of human ST2 (SEQ ID NO:328) fused at itsC-terminus to the N-terminus of a mouse IgG2a Fc region (SEQ ID NO:333).The third IL33 antagonist (hST2-hIL1RAcP-mFc, SEQ ID NO: 325) consistsof an in-line fusion having human ST2 (SEQ ID NO:328) at its N-terminus,followed by the extracellular region of human IL-1RAcP (SEQ ID NO:330),followed by a mouse IgG2a Fc (SEQ ID NO:333) at its C-terminus. Thefourth IL33 antagonist (mST2-mIL1RAcP-mFc, SEQ ID NO: 326) consists ofan in-line fusion having mouse ST2 (SEQ ID NO:329) at its N-terminus,followed by the extracellular region of mouse IL-1RAcP (SEQ ID NO:331),followed by a mouse IgG2a Fc (SEQ ID NO:333) at its C-terminus. Thefifth IL33 antagonist (hST2-hIL1RAcP-hFc, SEQ ID NO:327) consists of anin line fusion having human ST2 of SEQ ID NO: 328 at its N-terminus,followed by the extracellular region of human IL-1RAcP (SEQ ID NO: 330)followed by a human IgG1 Fc (SEQ ID NO: 332) at its C terminus. See,Table 2.

TABLE 2 Summary of IL33 Antagonists and the Component Parts Amino AcidSequence of Full Antagonist IL33 Antagonist Molecule D1 Component D2Component M Component hST2-hFc SEQ ID NO: 323 human ST2 Absent humanIgG1 Fc extracellular (SEQ ID NO: 332) (SEQ ID NO: 328) hST2-mFc SEQ IDNO: 324 human ST2 Absent mouse IgG2a Fc extracellular (SEQ ID NO: 333)(SEQ ID NO: 328) hST2-hIL1RAcP- SEQ ID NO: 325 human ST2 human IL-1RAcPmouse IgG2a Fc mFc extracellular extracellular (SEQ ID NO: 333) (SEQ IDNO: 328) (SEQ ID NO: 330) mST2-mIL1RAcP- SEQ ID NO: 326 mouse ST2 mouseIL-1RAcP mouse IgG2a Fc mFc extracellular extracellular (SEQ ID NO: 333)(SEQ ID NO: 329) (SEQ ID NO: 331) hST2-hIL1RAcP- SEQ ID NO: 327 humanST2 human IL-1RAcP human IgG1 Fc hFc extracellular extracellular (SEQ IDNO: 332) (SEQ ID NO: 328) (SEQ ID NO: 330)

The IL33 traps may comprise at least one multimerizing domain (sometimesreferred to herein by the abbreviation “M,” “M1,” “M2,” etc.). Ingeneral terms, the multimerizing domain(s) function to connect thevarious components of the IL33 antagonists (e.g., the IL33-bindingdomain(s)) with one another. A multimerizing domain may comprise anymacromolecule that has the ability to associate (covalently ornon-covalently) with a second macromolecule of the same or similarstructure or constitution. For example, a multimerizing domain maycomprise a polypeptide comprising an immunoglobulin CH3 domain. Anon-limiting example of a multimerizing domain is an Fc portion of animmunoglobulin, e.g., an Fc domain of an IgG selected from the isotypesIgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotypegroup.

Non-limiting exemplary multimerizing domains that can be used in theIL33 traps include human IgG1 Fc (SEQ ID NO: 332) or mouse IgG2a Fc (SEQID NO: 333). IL33 traps may comprise M components having an amino acidsequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to any of the exemplary M component aminoacid sequences set forth herein (e.g., SEQ ID NOs: 332 or 333).

In some embodiments, the IL33 traps comprise two multimerizing domains,M1 and M2, wherein M1 and M2 are identical to one another. For example,M1 can be an Fc domain having a particular amino acid sequence, and M2is an Fc domain with the same amino acid sequence as M1. The individualcomponents of the IL33 antagonists (e.g., D1, D2, M, etc.) can bearranged relative to one another in a variety of ways. Non-limitingexamples of all of the above noted arrangements, including an example ofan IL33 trap comprising two multimerizing domains (M1 and M2) and fourIL33 binding domains (D1, D2, D3 and D4,) are illustrated schematicallyin FIG. 1 .

The individual components of the IL33 traps (e.g., D1, D2, M1, M2, etc.)may be attached to one another directly (e.g., D1 and/or D2 may bedirectly attached to M, etc.); alternatively, the individual componentsmay be attached to one another via a linker component (e.g., D1 and/orD2 may be attached to M via a linker oriented between the individualcomponents; D1 may be attached to D2 via a linker; etc.).

Polypeptides that bind IL33 and/or its receptor (ST2 and/or IL-1 RAcP)and block ligand-receptor interaction are considered as IL33 antagonistsand are disclosed in PCT Publ. No. WO 2014/152195, which is incorporatedby reference in its entirety. The biological characteristics of the IL33traps are described in U.S. Publ. No. 2014/0271642, which isincorporated by reference herein in their entirety.

Other agents that may act as IL33 antagonists and which may be used inthe methods include immunoadhesins, peptibodies, and soluble ST2, orderivatives thereof; anti-IL33 receptor antibodies (e.g., anti-ST2antibodies, for example, AMG-282 (Amgen) or STLM15 (Janssen) or any ofthe anti-ST2 antibodies described in PCT Publ. Nos. WO 2012/113813, WO2013/173761, and WO 2013/165894, as well as U.S. Pat. Nos. 8,444,987 and7,452,980, which are each incorporated herein by reference in theirentirety. Other IL33 antagonists include ST2-Fc proteins, such as thosedescribed in PCT Publ. Nos. WO 2013/173761 and WO 2013/165894, which areeach incorporated herein by reference in their entirety.

In any of the methods described or exemplified herein, an IL-4Rantagonist may be administered as part of a therapeutic regimen. TheIL-4R antagonist is preferably administered in combination with the IL33antagonist, though the IL-4R antagonist need not be administered at thesame time as the IL33 antagonist. An IL-4R antagonist may comprise anyagent that binds to or interacts with IL-4Rα or an IL-4R ligand, andinhibits or attenuates the normal biological signaling function of atype 1 and/or a type 2 IL-4 receptor. The IL-4R may comprise the aminoacid sequence of SEQ ID NO: 347, or a biologically active fragmentthereof. A type 1 IL-4 receptor is a dimeric receptor comprising anIL-4Rα chain and a γc chain. A type 2 IL-4 receptor is a dimericreceptor comprising an IL-4Rα chain and an IL-13Rα1 chain. Type 1 IL-4receptors interact with and are stimulated by IL-4, while type 2 IL-4receptors interact with and are stimulated by both IL-4 and IL-13. Thus,the IL-4R antagonists used in the methods may function by blockingIL-4-mediated signaling, IL-13-mediated signaling, or both IL-4- andIL-13-mediated signaling. The IL-4R antagonists may thus inhibit orprevent the interaction of IL-4 and/or IL-13 with a type 1 or type 2receptor.

Non-limiting examples of categories of IL-4R antagonists include smallmolecule IL-4R antagonists, nucleic acid-based inhibitors of IL-4Rexpression or activity (e.g., siRNA or antisense), peptide-basedmolecules that specifically interact with IL-4R (e.g., peptibodies),“receptor-bodies” (e.g., engineered molecules comprising theligand-binding domain of an IL-4R component), IL-4R-binding scaffoldmolecules (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins,tetratricopeptide repeat proteins, fibronectin-based scaffoldconstructs, and other scaffolds based on naturally occurring repeatproteins, and anti-IL-4R aptamers or portions thereof.

In preferred embodiments, an IL-4R antagonist comprises an antibody thatspecifically binds to human IL-4R. Antibodies are typically referred toherein according to the following nomenclature: Fc prefix (e.g. “H1M,”or “H4H”), followed by a numerical identifier (e.g., “9559,” “9566,” or“9629” as shown in Table 1), followed by a “P,” or “N” suffix. Accordingto this nomenclature, an antibody may be referred to herein as, e.g.,“H1M9559N,” “H1M9566N,” “H4H9629P,” etc. The H1M and H4H prefixes on theantibody designations used herein indicate the particular Fc regionisotype of the antibody. For example, an “H1M” antibody has a mouse IgG1Fc, whereas an “H4H” antibody has a human IgG4 Fc. An antibody having aparticular Fc isotype can be converted to an antibody with a differentFc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted toan antibody with a human IgG4, etc.), but in any event, the variabledomains (including the CDRs)—which are indicated by the numericalidentifiers shown in Table 1—will remain the same, and the bindingproperties are expected to be identical or substantially similarregardless of the nature of the Fc

In preferred embodiments, the anti-IL-4R antibody is dupilumab. See U.S.Pat. Nos. 7,605,237, 7,608,693, and 9,290,574, which are incorporated byreference.

Human anti-IL-4R antibodies can be generated as described in U.S. Pat.No. 7,608,693. One exemplary IL-4R antibody is a mouse antibody specificfor mouse IL-4R, and has the following amino acid sequences: a heavychain variable region (HCVR) comprising SEQ ID NO: 335 and a light chainvariable domain (LCVR) comprising SEQ ID NO: 336. The human anti-IL-4Rantibody, referred to as dupilumab, specifically binds to human IL-4Rαand comprises a heavy chain variable region (HCVR) comprising SEQ ID NO:337 and a light chain variable region (LCVR) comprising SEQ ID NO: 338,a heavy chain complementarity determining region 1 (HCDR1) comprisingSEQ ID NO: 339, a HCDR2 comprising SEQ ID NO: 340, a HCDR3 comprisingSEQ ID NO: 341, a light chain complementarity determining region 1(LCDR1) comprising SEQ ID NO: 342, a LCDR2 comprising SEQ ID NO: 343 anda LCDR3 comprising SEQ ID NO: 344. The full-length heavy chain ofdupilumab is shown as SEQ ID NO: 345 and the full length light chain isshown as SEQ ID NO: 346.

In some embodiments, the IL-4R antagonist comprises an anti-IL-4Rαantibody, or antigen-binding fragment thereof comprising a heavy chainvariable region (HCVR), light chain variable region (LCVR), and/orcomplementarity determining regions (CDRs) comprising any of the aminoacid sequences of the anti-IL-4R antibodies as set forth in U.S. Pat.Nos. 7,605,237 and 7,608,693. In some embodiments, the IL-4R antagonistcomprises an anti-IL-4R antibody having the binding characteristics ofthe reference antibody referred to herein as dupilumab (U.S. Pat. No.7,605,237 and U.S. Pat. No. 7,608,693). In some embodiments, theanti-IL-4Rα antibody or antigen-binding fragment thereof comprises theheavy chain complementarity determining regions (HCDRs) of a heavy chainvariable region (HCVR) comprising the amino acid sequence of SEQ ID NO:337 and the light chain complementarity determining regions (LCDRs) of alight chain variable region (LCVR) comprising the amino acid sequence ofSEQ ID NO: 338. In some embodiments, the anti-IL-4Rα antibody orantigen-binding fragment thereof comprises three HCDRs (HCDR1, HCDR2 andHCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein the HCDR1comprises the amino acid sequence of SEQ ID NO: 339; the HCDR2 comprisesthe amino acid sequence of SEQ ID NO: 340; the HCDR3 comprises the aminoacid sequence of SEQ ID NO: 341; the LCDR1 comprises the amino acidsequence of SEQ ID NO: 342; the LCDR2 comprises the amino acid sequenceof SEQ ID NO: 343; and the LCDR3 comprises the amino acid sequence ofSEQ ID NO: 344. In yet other embodiments, the anti-IL-4R antibody orantigen-binding fragment thereof comprises an HCVR comprising SEQ ID NO:337 and an LCVR comprising SEQ ID NO: 338. In yet other embodiments, theanti-IL-4R antibody or antigen-binding fragment thereof comprises anHCVR comprising SEQ ID NO: 335 and an LCVR comprising SEQ ID NO: 336. Insome embodiments, the anti-IL-4R antibody or antigen-binding fragmentthereof comprises a heavy chain (HC) amino acid sequence as set forth inSEQ ID NO: 345 and a light chain (LC) amino acid sequence as set forthin SEQ ID NO: 346.

In some embodiments, the IL-4R antibody or antigen-binding fragmentthereof comprises the heavy chain complementarity determining regions(HCDRs) of a heavy chain variable region (HCVR) comprising the aminoacid sequence of SEQ ID NO:335 or SEQ ID NO: 337 and the light chaincomplementarity determining regions (LCDRs) of a light chain variableregion (LCVR) comprising the amino acid sequence of SEQ ID NO:336 or SEQID NO: 338.

In some embodiments, the IL-4R antibody or antigen-binding fragmentthereof comprises three HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs(LCDR1, LCDR2 and LCDR3), wherein the HCDR1 comprises the amino acidsequence of SEQ ID NO: 339, the HCDR2 comprises the amino acid sequenceof SEQ ID NO:340; the HCDR3 comprises the amino acid sequence of SEQ IDNO:341; the LCDR1 comprises the amino acid sequence of SEQ ID NO:342;the LCDR2 comprises the amino acid sequence of SEQ ID NO:343; and theLCDR3 comprises the amino acid sequence of SEQ ID NO:344.

In some embodiments, the IL-4R antibody or antigen-binding fragmentthereof for use in the methods of the disclosure comprises an HCVRcomprising the amino acid sequence of SEQ ID NO: 335 or SEQ ID NO: 337and an LCVR comprising the amino acid sequence of SEQ ID NO: 336 or SEQID NO: 338.

In some embodiments, the IL-4R antibody or antigen-binding fragmentthereof for use in the methods of the disclosure comprises an HCVR/LCVRamino acid sequence pair of SEQ ID NOs: 335/336 or SEQ ID NOs: 337/338.

Other anti-IL-4Rα antibodies include, for example, the antibody referredto and known in the art as AMG317 (Corren et al., 2010, Am J. RespirCrit Care Med., 181(8):788-796), or MEDI 9314, or any of the anti-IL-4Rαantibodies as set forth in any of U.S. Pat. Nos. 7,186,809, 7,605,237,7,638,606, 8,092,804, 8,679,487, or 8,877,189.

The anti-IL-4Rα and the IL33 antibodies may have pH-dependent bindingcharacteristics. For example, an anti-IL-4α antibody or an anti-IL33antibody may exhibit reduced binding to IL-4Rα, or to IL33,respectively, at acidic pH as compared to neutral pH. Alternatively, ananti-IL-4Rα antibody or an anti-IL33 antibody may exhibit enhancedbinding to its antigen at acidic pH as compared to neutral pH. An“acidic pH” includes pH values less than about 6.2, e.g., about 6.0,5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35,5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. A “neutral pH” includes apH of about 7.0 to about 7.4, as well as about 7.0, 7.05, 7.1, 7.15,7.2, 7.25, 7.3, 7.35, and 7.4.

In another aspect, an IL33 antagonist is used alone or in combinationwith an IL-4R antagonist for treating or inhibiting an inflammatorycondition of the lungs. The IL33 antagonist or combination may be usedfor treating or inhibiting one or more of asthma, COPD, or ACOS. TheIL33 antagonist or combination may be used for treating or inhibitingnasal polyps. The combination may be used for treating or inhibiting oneor more of eosinophilic asthma, eosinophilic COPD, or eosinophilic ACOS.The IL33 antagonist or combination may be used for treating orinhibiting one or more of the high eosinophilic subset of eosinophilicasthma, eosinophilic COPD, or eosinophilic ACOS. The combinationdemonstrates enhanced efficacy, as compared to the treatment orinhibition obtained when each antibody is used alone as monotherapy.

In some embodiments, an IL33 antagonist or a combination of an IL33antagonist and an IL-4R antagonist is used in the manufacture of amedicament for the treatment or inhibition of any one of eosinophilicasthma, eosinophilic Chronic Obstructive Pulmonary Disease (COPD),eosinophilic asthma-Chronic Obstructive Pulmonary Disease overlapsyndrome (ACOS), high-eosinophil eosinophilic asthma, high-eosinophileosinophilic COPD, high-eosinophil eosinophilic ACOS, or nasal polyps.In preferred embodiments, the IL33 antagonist or combination is used inthe manufacture of a medicament for such treatment or inhibition of anyone of eosinophilic asthma, eosinophilic COPD, eosinophilic ACOS,high-eosinophil eosinophilic asthma, high-eosinophil eosinophilic COPD,high-eosinophil eosinophilic ACOS, or nasal polyps when a patientthereof has one or more risk alleles associated with eosinophilicasthma, eosinophilic COPD, or eosinophilic ACOS in the intronic IL1RL1variant rs1420101 (SEQ ID NO: 357) or variant in linkage disequilibriumthereof, in the IL33 variant rs1342326 (SEQ ID NO: 358) or variant inlinkage disequilibrium thereof, or in both the intronic IL1RL1 variantrs1420101 (SEQ ID NO: 357) or variant in linkage disequilibrium thereofand the IL33 variant rs1342326 (SEQ ID NO: 358) or variant in linkagedisequilibrium thereof.

According to such use, the IL33 antagonist may comprise an IL33 trap.The IL33 trap may comprise a first IL33 binding domain comprising anIL33 binding portion of IL1RL1 and a second IL33 binding domaincomprising an extracellular portion of IL-1RAcP. The IL33 antagonist mayalternatively comprise an antibody or antigen-binding fragment thereofthat specifically binds to IL33. The antibody or antigen-bindingfragment thereof that specifically binds to IL33 may comprise the H1,H2, and H3 domains of SEQ ID NO: 274 and the L1, L2, and L3 domains ofSEQ ID NO: 282. The IL-4R antagonist may comprise an antibody orantigen-binding fragment thereof that specifically binds to IL-4R. Theantibody or antigen-binding fragment thereof that specifically binds toIL-4R may comprise the H1, H2, and H3 domains of SEQ ID NO: 337 and theL1, L2, and L3 domains of SEQ ID NO: 338.

The following examples are provided to describe the disclosure ingreater detail. They are intended to illustrate, not to limit, thedisclosure.

EXAMPLE 1 Anti-IL33 Antibody, Anti-IL-4R Antibody, and a Combination ofBoth in a Chronic House Dust Mite-Induced Fibrosis and Severe LungInflammation Model

Chronic inflammatory airway diseases are a consequence of recurrentepisodes of airway inflammation predominantly due to repeated exposureto allergens or other pathogens. In humans, such chronic insults inducea vast array of pathologies that include pulmonary infiltration byimmune cells, increased cytokine production, mucus production andcollagen deposition. This increase in inflammatory cytokines and immunecell infiltrates, accompanied by intense airway remodeling leads toairway narrowing, hyperresponsiveness to inhaled triggers such asallergens or pathogens, airway obstruction and loss of lung function.

To determine the effect of anti-IL33 inhibition in a relevant in vivomodel, a chronic house dust mite extract (HDM)-induced fibrosis andsevere lung inflammation and remodeling study was conducted in mice thatwere homozygous for the expression of human IL33 in place of mouse IL33(IL33 HumIn mice). See U.S. Publ. Nos. 2015/0320021 and 2015/0320022.Chronic HDM extract exposure induces severe lung inflammation, resultingin significant cellular infiltrate, cytokine expression, and remodeling.Efficacy of an anti-IL33 antibody, an anti-mouse IL-4Rα antibody or acombination of both was compared in this model. The anti-mouse IL-4Rαantibody used in this study is designated M1M1875N and comprises theHCVR/LCVR amino acid sequence pair of SEQ ID NOs: 335/336. The anti-IL33antibody used in this study is designated H4H9675P and comprises theHCVR/LCVR amino acid sequence pair of SEQ ID NOs: 274/282.

IL33 HumIn mice were intranasally administered either 50 μg house dustmite extract (HDM; Greer, #XPB70D3A2.5) diluted in 20 μL of 1× phosphatebuffered saline (PBS), or 20 μL of 1×PBS for 3 days per week for 15weeks. A second control group of IL33 HumIn mice were administered 50 μgHDM extract diluted in 20 μL of 1×PBS for 3 days per week for 11 weeks,to assess the severity of the disease at the onset of antibodytreatment. Four groups of HDM challenged mice were injectedsubcutaneously with 25 mg/kg of either the anti-IL33 antibody H4H9675P,the anti-mouse IL-4Rα antibody M1M1875N, a combination of bothantibodies, or an isotype control antibody starting after 11 weeks ofHDM challenge and then twice per week until the end of the HDM challenge(4 weeks of antibody treatment). On day 108 of the study, all mice weresacrificed and their lungs were harvested. Experimental dosing andtreatment protocol for groups of mice are shown in Table 3.

TABLE 3 Experimental dosing and treatment protocol for groups of miceLength of Intranasal intranasal Group Mice challenge challenge Antibody1 IL33 HumIn 1X PBS 15 weeks None mice 2 IL33 HumIn 50 μg HDM in 11weeks None mice 20 μL 1X PBS 3 IL33 HumIn 50 μg HDM in 15 weeks Nonemice 20 μL 1X PBS 4 IL33 HumIn 50 μg HDM in 15 weeks Isotype controlmice 20 μL 1X PBS antibody 5 IL33 HumIn 50 μg HDM in 15 weeks Anti-IL33antibody mice 20 μL 1X PBS (H4H9675P) 6 IL33 HumIn 50 μg HDM in 15 weeksAnti-IL-4Rα mice 20 μL 1X PBS antibody (M1M1875N) 7 IL33 HumIn 50 μg HDMin 15 weeks Anti-IL33 mice 20 μL 1X PBS (H4H9675P) antibody +Anti-IL-4Rα (M1M1875N) antibody

Lung harvest for cytokine analysis. Elevated lung levels of keymediators such as the prototypic type 2 cytokines IL-4, IL-5, and IL-13,as well as cytokines more characteristic of type 1 immune responses,such as IL-1β or TNFα have been involved in human the development oflung diseases. Lung levels of these inflammatory cytokines were measuredin the study.

After exsanguination, the cranial and middle lobes of the right lungfrom each mouse were removed and placed into tubes containing a solutionof tissue protein extraction reagent (1×T-PER reagent; Pierce, #78510)supplemented with 1× Halt Protease inhibitor cocktail (ThermoScientific, #87786). All further steps were performed on ice. The volumeof T-PER Reagent (containing the protease inhibitor cocktail) wasadjusted for each sample to match a 1:7 (w/v) tissue to T-PER ratio.Lung samples were mechanically disrupted using the TissueLyser II(Qiagen #85300). The resulting lysates were centrifuged to pelletdebris. The supernatants containing the soluble protein extracts weretransferred to fresh tubes and stored at 4° C. until further analysis.

Total protein content in the lung protein extracts was measured using aBradford assay. For the assay, 10 μL of diluted extract samples wereplated into 96 well plates in duplicates and mixed with 200 μL of 1× DyeReagent (Biorad, #500-0006). Serial dilutions of bovine serum albumin(BSA; Sigma, #A7979), starting at 700 μg/mL in 1×T-Per reagent were usedas a standard to determine the protein concentration of the extracts.After a 5-minute incubation at room temperature, absorbance at 595 nmwas measured on a Molecular Devices SPECTRAMAX® M5 plate reader. Dataanalysis to determine total lung extract protein content based on theBSA standard was performed using GraphPad Prism™ software.

Cytokine concentrations in the lung protein extracts were measured usinga Proinflammatory Panel 1 (mouse) multiplex immunoassay kit (MesoScaleDiscovery, # K15048G-2) and a custom mouse 6plex MULTI-SPOT® immunoassaykit (MesoScale Discovery, #K152A41-4), according to the manufacturer'sinstructions. Briefly, 50 μL/well of calibrators and samples (diluted inDiluent 41) were added to plates pre-coated with capture antibodies andincubated at room temperature while shaking at 700 rpm for 2 hours. Theplates were then washed 3 times with 1×PBS containing 0.05% (w/v) TWEEN®-20 surfactant, followed by the addition of 25 μL of Detection AntibodySolution diluted in Diluent 45. After a 2 hour incubation at roomtemperature while shaking, the plate was washed 3 times, and 150 μL of2× Read Buffer was added to each well. Electrochemiluminescence wasimmediately read on a MSD Spector instrument. Data analysis wasperformed using GraphPad Prism software.

Each cytokine concentration in lung total protein extracts from all micein each group was normalized to the total protein content of theextracts measured by the Bradford assay, and expressed for each group asaverage pg of cytokine per mg of total lung proteins (pg/mg lungprotein, ±SD) as shown in Table 4.

Lung cytokines analysis. As shown in Table 4, the level of the cytokinesand chemokines IL-4, IL-5, IL-6, IL-1β and MCP-1 released in the lungsof IL33 HumIn mice receiving HDM for 15 weeks, with or without treatmentwith an isotype control antibody were significantly higher than in IL33HumIn mice challenged with 1×PBS alone. Similarly, there was a trendtowards an increased release of the cytokines IL-13 and TNFα in thelungs of IL33 HumIn mice receiving HDM for 15 weeks. In contrast, therewas a significant reduction in the levels of IL-6, IL-13 and MCP-1 inthe lungs of IL33 HumIn mice treated with a combination of anti-IL33 andanti-mouse IL-4Rα antibodies during the last four weeks of the chronicHDM challenge as compared to IL33 HumIn mice administered HDM with anisotype control antibody during this time period. There was a trendtowards reduced IL-4, IL-5, IL-1β and TNFα lung levels in IL33 HumInmice treated with a combination of anti-IL33 and anti-mouse IL-4Rαantibodies during the last four weeks of the chronic HDM challenge ascompared to IL33 HumIn mice administered HDM with an isotype controlantibody during this time period. The effects on lung cytokines observedwith the combination anti-IL33 and anti-mouse IL-4Rα antibodies wasgreater than treatment with either individual antibodies alone.

TABLE 4 Cytokine concentration in lung protein extracts Mean Mean MeanMean Mean Mean Mean [IL-4] [IL-5] [IL-13] [IL-6] [IL-1β] [TNFα] [MCP-1]in lung in lung in lung in lung in lung in lung in lung protein proteinprotein protein protein protein protein extracts extracts extractsextracts extracts extracts extracts (pg/mg (pg/mg (pg/mg (pg/mg (pg/mg(pg/mg (pg/mg lung lung lung lung lung lung lung Experimental protein)protein) protein) protein) protein) protein) protein) group (±SD) (±SD)(±SD) (±SD) (±SD) (±SD) (±SD) 1. 1X PBS challenge 0.13 0.80 ND   4.75  1.97  2.86  4.12 (n = 5) (±0.17)  (±1.41)   (±3.39)  (±1.67) (±1.01)(±1.12) 2. HDM challenge 5.71 7.31 0.20 293.1 181.8 17.39 43.06 11 weeks(n = 4)  (±3.76) * (±3.67)  (±0.03)   (±139.3) *  (±131.0) * (±8.90)(±24.21)  3. HDM challenge 2.70 5.13 0.19 308.3  51.79 15.38 105.6  15weeks (n = 4) (±1.71)  (±3.20)  (±0.03)  (±390.1)   (±16.97) (±8.11)(±106.5) *  4. HDM challenge 5.46 7.00 0.22 395.0 162.3 19.57 141.7  15weeks + isotype  (±3.38) **  (±4.50) * (±0.02)   (±270.1) **  (±166.5)** (±14.81)  (±126.3) **  control antibody (n = 4) 5. HDM challenge 1.151.93 0.20 136.8 122.9 17.05 16.64 15 weeks + anti- (±1.38)  (±1.90) (±0.02)  (±164.1)  (±194.1)   (±4.48) * (±6.40) IL33 antibody (n = 5) 6.HDM challenge 2.88 13.13  0.16  18.24  26.73  7.85 11.63 15 weeks +anti- (±2.43)   (±12.81) ** (±0.03)   (±12.43)  (±20.94) (±4.89) (±8.69)mouse IL-4Rα antibody (n = 5) 7. HDM challenge 0.47 0.73 0.10   7.46  3.722  3.07  4.62 15 weeks + anti- (±0.13)  (±0.37)   (±0.05) ^(††)    (±2.52) ^(†)  (±1.59) (±1.34)  (±1.27) ^(††) IL33 + anti-mouseIL-4Rα antibodies (n = 5) Note: Statistical significance determined byKruskal-Wallis One-way ANOVA with Dunn's multiple comparison post-hoctest is indicated (* = p < 0.05, ** = p < 0.01, compared to groups 1:IL33 HumIn mice, Saline challenge; ^(†) p < 0.05, ^(††) p < 0.01,compared to group 4: IL33 Humin mice, HDM challenge 15 weeks + Isotypecontrol antibody). ND: Not determined.

Lung harvest for gene expression analysis. After exsanguination, theaccessory lobe of the right lung from each mouse was removed, placedinto tubes containing 400 μL of RNA Later (Ambion, #AM7020) and storedat −20° C. until processing. Tissues were homogenized in TRIzol andchloroform was used for phase separation. The aqueous phase, containingtotal RNA, was purified using MagMAX™-96 for Microarrays Total RNAIsolation Kit (Ambion by Life Technologies, #AM1839) according tomanufacturer's specifications. Genomic DNA was removed usingMagMAX™Turbo™DNase Buffer and TURBO DNase from the MagMAX kit listedabove. mRNA (up to 2.5 μg) was reverse-transcribed into cDNA usingSuperScript® VILO™ Master Mix (Invitrogen by Life Technologies,#11755500). cDNA was diluted to 2 nd/μL and 10 ng cDNA was amplifiedwith the TaqMan® Gene Expression Master Mix (Applied Biosystems by LifeTechnologies, #4369542) and the relevant probes (Life Technologies;mouse B2m: Mm00437762_m1; mouse Il4: Mm00445259_m1; mouse Il5:Mm00439646_m1; mouse Il13: Mm00434204_m1; mouse Il9: Mm00434305_m1;mouse Il6: Mm00446190_m1; mouse Ccl2: Mm00441242_m1; mouse Ccl11:Mm00441238_m1; mouse Ccl24: Mm00444701_m1; mouse Tnf: Mm00443258_m1;mouse Tgfb1: Mm01178820_m1; mouse Il1rl1: Mm00516117_m1; mouse Il13ra2:Mm00515166_m1; mouse Col15a1: Mm00456584_m1; mouse Col24a1:Mm01323744_m1;) using the ABI 7900HT Sequence Detection System (AppliedBiosystems). B2m was used as the internal control genes to normalize anycDNA input differences. The reference group used for normalization ofall samples was the average of Group 1 samples (‘1X PBS Challenge’).Expression of each gene was normalized to B2m expression within the samesample and expressed relative to its normalized expression in thereference group (mean ±SD), as shown in Table 5.

Lung gene expression analysis. As shown in Table 5, the level ofexpression of the cytokines, chemokines and collagen genes Il4, Il13,Il6, Ccl2, Tgfb1, Il13ra2 and Col24a1 in the lungs of IL33 HumIn micereceiving HDM for 15 weeks, with or without treatment with an isotypecontrol antibody were significantly increased compared to IL33 HumInmice challenged with 1×PBS alone. Similarly, there was a trend towardsan increase in expression of the genes Il5, Il9, Ccl11, Ccl24, Tnf,Il1rl1 and Col15a1 in the lungs of IL33 HumIn mice receiving HDM for 15weeks.

In contrast, there was a significant reduction in the expression levelsof Il6, Ccl2, Ccl11 and Ccl24 in the lungs of IL33 HumIn mice treatedwith a combination of anti-IL33 and anti-mouse IL-4Rα antibodies duringthe last four weeks of the chronic HDM challenge as compared to IL33HumIn mice administered HDM with an isotype control antibody during thistime period. There was a trend towards reduced Il4, Il5, Il13, Il9, Tnf,Tgfb1, Il1rl1, Il13ra2, Col15a1 and Col24a1 expression levels in micetreated with a combination of anti-IL33 and anti-mouse IL-4Rα antibodiesduring the last four weeks of the chronic HDM challenge as compared toIL33 HumIn mice administered HDM with an isotype control antibody duringthis time period. The effects on gene expression observed with thecombination anti-IL33 and anti-mouse IL-4Rα antibodies was greater thantreatment with either individual antibodies alone.

TABLE 5 Gene expression (TaqMan) in mouse lungs. Mean Mean Mean MeanMean Mean Mean Mean Relative Relative Relative Relative RelativeRelative Relative Relative Il4 Il5 Il13 Il9 Il6 Ccl2 Ccl11 Ccl24expression expression expression expression expression expressionexpression expression Experimental in lung in lung in lung in lung inlung in lung in lung in lung group (±SD) (±SD) (±SD) (±SD) (±SD) (±SD)(±SD) (±SD) 1. 1X PBS challenge 1.03 1.54  4.51 15.91 1.25 1.20  1.241.05 (n = 5) (±0.28)  (±1.61) (±7.59) (±34.81) (±1.09)  (±0.93)  (±1.07)(±0.33)  2. HDM challenge 12.78  7.13 114.1  38.66 9.12 18.86  13.3615.44  11 weeks (n = 4)  (±8.45) * (±3.49) (±68.3) *  (±30.04) (±1.65) (±8.40)  (±5.05) (±12.02)  3. HDM challenge 6.27 4.20 58.05 30.63 8.9222.61   8.65 4.58 15 weeks (n = 4) (±3.39)  (±1.51) (±31.61)  (±20.54)(±4.55)  (±13.37) *  (±3.20) (±1.91)  4. HDM challenge 10.98  5.50 92.5119.51 13.80  24.53  12.14 12.41  15 weeks + isotype  (±5.46) * (±3.16) (±75.96) * (±10.29)  (±6.98) **  (±9.13) ** (±7.82) (±8.73)  controlantibody (n = 4) 5. HDM challenge 2.80 1.74 12.91 0.00 3.87 5.20  6.211.45 15 weeks + anti- (±3.11)  (±1.11) (±12.93)  (±0.00) (±3.00) (±2.44)  (±3.55) (±2.09)  IL33 antibody (n = 5) 6. HDM challenge 1.877.98 69.56 63.50 2.77 2.97  1.00 0.44 15 weeks + anti- (±1.03)  (±6.52) (±66.86) * (±92.04) (±1.39)  (±1.86)  (±0.18) (±0.34)  mouse IL-4Rαantibody (n = 5) 7. HDM challenge 1.37 1.56  9.34 0.57 1.04 1.08  0.720.15 15 weeks + anti- (±0.35)  (±0.97) (±3.10) (±1.27)  (±0.31) ^(††)  (±0.24) ^(†† §)   (±0.28) ^(†)  (±0.10) ^(††) IL33 + anti-mouse IL-4Rαantibodies (n = 5) Mean Mean Mean Mean Mean Mean Relative RelativeRelative Relative Relative Relative Tnf Tgfb1 Il1rl1 Il13rα2 Col15α1Col24α1 expression expression expression expression expressionexpression Experimental in lung in lung in lung in lung in lung in lunggroup (±SD) (±SD) (±SD) (±SD) (±SD) (±SD) 1. 1X PBS challenge 1.02 1.001.11 1.59 1.00  1.02 (n = 5) (±0.24)  (±0.11)  (±0.58)  (±1.96) (±0.10) (±0.16) 2. HDM challenge 1.45 1.40 3.03 48.43 2.75 24.55 11 weeks (n =4) (±0.41)  (±0.27)   (±0.88) * (±34.21) (±0.96)    (±7.97) ** 3. HDMchallenge 1.58 1.32 2.53 32.07 3.00 17.25 15 weeks (n = 4) (±0.43) (±0.33)   (±0.79) * (±13.45) (±1.22)   (±5.29) * 4. HDM challenge 1.591.37 3.45 52.02 3.80 23.58 15 weeks + isotype (±0.78)   (±0.12) * (±1.48) * (±40.63)  (±0.96) *     (±6.18) *** control antibody (n = 4)5. HDM challenge 1.38 1.22 0.99 13.54 1.64 10.58 15 weeks + anti-(±0.27)  (±0.24)  (±0.47)  (±12.25) (±0.30)  (±5.42) IL33 antibody (n =5) 6. HDM challenge 1.00 1.13 3.38 1.89 1.24  7.08 15 weeks + anti-(±0.25)  (±0.20)  (±1.97)  (±0.59) (±0.28)  (±4.56) mouse IL-4Rαantibody (n = 5) 7. HDM challenge 0.68 1.09 1.12 1.89 0.74  1.76 15weeks + anti-  (±0.08) ^(§) (±0.12)  (±0.57)  (±0.27)   (±0.21) ^(†† §)  (±0.15) ^(†) IL33 + anti-mouse IL-4Rα antibodies (n = 5) Note:Statistical significance determined by Kruskal-Wallis One-way ANOVA withDunn's multiple comparison post-hoc test is indicated (* = p < 0.05, **= p < 0.01, *** = p < 0.01 compared to groups 1: IL33 HumIn mice, Salinechallenge; ^(§) p < 0.05, ^(§§)p < 0.01, compared to group 3: IL33 Huminmice, HDM challenge 15 weeks; ^(†) p < 0.05, ^(††) p < 0.01, compared togroup 4: IL33 Humin mice, HDM challenge 15 weeks + Isotype controlantibody).

Lung harvest for pulmonary cell infiltrate analysis. Pulmonaryinfiltration by immune cells is observed in multiple airway inflammatorydiseases, including asthma and COPD. Neutrophilic lung inflammation hasbeen associated with lower lung function and severe tissue remodeling inasthma patients. Eosinophilic lung inflammation is a hallmark of type 2inflammation usually seen in atopic diseases. In humans, high CD4/CD8ratios are observed in patients with granulomatous lung diseases andother chronic inflammatory conditions. Flow cytometry was used in thestudy to determine the level of cellular infiltration in the lungs ofHDM-exposed mice.

After exsanguination, the caudal lobe of the right lung from each mousewas removed, chopped into cubes that were approximately 2 to 3 mm insize, and then placed into a tube containing a solution of 20 μg/mLDNAse (Roche, #10104159001) and 0.7 U/mL Liberase TH (Roche,#05401151001) diluted in Hank's Balanced Salt Solution (HBSS) (Gibco,#14025), which was incubated in a 37′ C. water bath for 20 minutes andvortexed every 5 minutes. The reaction was stopped by addingethylenediaminetetraacetic acid (EDTA, Gibco, #15575) at a finalconcentration of 10 mM. Each lung was subsequently dissociated using agentleMACS dissociator (Miltenyi Biotec, #130-095-937), then filteredthrough a 70 μm filter and centrifuged. The resulting lung pellet wasresuspended in 1 mL of 1× red blood cell lysing buffer (Sigma, #R7757)to remove red blood cells. After incubation for 3 minutes at roomtemperature, 3 mL of 1×DMEM was added to deactivate the red blood celllysing buffer. The cell suspensions were then centrifuged, and theresulting cell pellets were resuspended in 5 mL of MACS buffer (autoMACSRunning Buffer; Miltenyi Biotec, #130-091-221). The resuspended sampleswere filtered through a 70 μm filter and 1×10⁶ cells per well wereplated in a 96-well V-bottom plate. Cells were then centrifuged and thepellets were washed in 1×PBS. After a second centrifugation, the cellpellets were resuspended in 100 μL of LIVE/DEAD Fixable Blue Dead CellStain (Life Technologies, # L23105) diluted at 1:500 in 1×PBS todetermine cell viability and incubated for 20 minutes at roomtemperature while protected from light. After one wash in 1×PBS, cellswere incubated in a solution of MACS buffer containing 10 μg/mL ofpurified rat anti-mouse CD16/CD32 Fc Block, (Clone: 2.4G2; BDBiosciences, #553142) for 10 minutes at 4° C. The cells were thenincubated in the appropriate 2× antibody mixture (described in Table 6)diluted in MACS buffer for 30 minutes at 4° C. while protected fromlight. After antibody incubation, the cells were washed twice in MACSbuffer, resuspended in BD CytoFix (BD Biosciences, #554655) and thenincubated for 15 minutes at 4° C. while protected from light. The cellswere subsequently washed, resuspended in MACS buffer, and thentransferred to BD FACS tubes (BD Biosciences, #352235) for analysis ofcellular infiltrates by flow cytometry.

CD4 and CD8 T cells were defined as cells that were live, CD45⁺,SSC^(Lo), FSC^(Lo), CD3⁺, CD19⁻, CD4⁺, CD8⁻ and live, CD45⁺, SSC^(Lo),FSC^(Lo), CD3⁺, CD19⁻, CD4⁻, CD8⁺ respectively. Activated CD4 T cellswere defined as cells that were live, CD45⁺, SSC^(Lo), FSC^(Lo), CD3⁺,CD19⁻, CD4⁺, CD8⁻, and CD69⁺. Activated CD8 T cells were defined ascells that were live, CD45⁺, SSC^(Lo), FSC^(Lo), CD3⁺, CD19⁻, CD4⁻,CD8⁺, and CD69⁺. Activated B cells were defined as cells that were live,CD45⁺, SSC^(Lo), FSC^(Lo), CD3⁻, CD19⁺, and CD69⁺. ST2+CD4+T cells weredefined as cells that were live, CD45⁺, SSC^(Lo), FSC^(Lo), CD3+, CD19−,ST2+ and CD4⁺. Eosinophils were defined as live, CD45⁺, GR1⁻,CD11c^(lo), SiglecF^(hi). Alveolar macrophages were defined as live,CD45⁺, GR1⁻, CD11c^(Hi), SiglecF^(hi). Data for activated cells isexpressed as frequency of activated cells (CD69⁺) within the parentpopulation (CD4, ±SD). Data for ST2+CD4+T cells is expressed asfrequency of T cells (defined as cells that were live, CD45⁺, SSC^(Lo),FSC^(Lo), CD3+ and CD19−). Data for Eosinophils and Alveolar macrophagesare expressed as frequency of live cells. CD4/CD8 T cells ratio iscalculated as the ratio of the frequency of CD4 T to the frequency ofCD8 T cells within the live population. All data are shown in Table 7.

TABLE 6 Antibodies Used for Flow Cytometry Analysis Catalogue FinalAntibody Fluorochrome Manufacturer Number dilution CD45.2 PerCP-Cy5.5eBioscience 45-0454 1/800 Siglec-F BV 421 BD 562681 1/200 F4/80 APCeBioscience 17-4801-82 1/200 Ly6G BUV395 BD 563978 1/200 Ly6C PE-Cy7 BD560593 1/100 CD11c PE eBioscience 12-0114-82 1/200 CD11b FITCeBioscience 53-0112-82 1/200 CD19 BV650 BD 562701 1/400 CD3 PE-Cy7 BD552774 1/200 CD4 BV421 BioLegend 100438 1/200 CD8 BUV 395 BD 5637861/400 NKp46 FITC eBioscience 11-3351 1/800 (CD335) CD69 PE eBioscience12-0691 1/200 CD25 BV510 BioLegend 102042 1/200 ST2 APC BioLegend 1453061/200

Pulmonary cell infiltrate analysis. As shown in Table 7, the frequencyof eosinophils, activated B cells, activated CD8 cells, ST2+Cd4+T cellsand CD4/CD8 T cells ratio in the lungs of IL33 HumIn mice receiving HDMfor 15 weeks, with or without treatment with an isotype control antibodywere significantly higher than in IL33 HumIn mice challenged with 1×PBSalone. Similarly, there was a trend towards an increased frequency ofactivated CD4 T cells in the lungs of IL33 HumIn mice receiving HDM for15 weeks. There was a trend towards a decreased frequency of alveolarmacrophages detected by flow cytometry in the lungs of IL33 HumIn micereceiving HDM for 15 weeks, in the absence or presence of an isotypecontrol antibody treatment. The frequency of alveolar macrophages wassignificantly increased in the lungs of IL33 HumIn mice treated with acombination of anti-IL33 and anti-mouse IL-4Rα antibodies during thelast four weeks of the chronic HDM challenge as compared to IL33 HumInmice administered HDM with an isotype control antibody during this timeperiod. Similarly, there was a trend towards reduced frequency ofeosinophils, activated CD4 and CD8 T cells, activated B cells, ST2+CD4+Tcells as well as CD4/CD8 T cells ratio in the lungs of mice treated witha combination of anti-IL33 and anti-mouse IL-4Rα antibodies during thelast four weeks of the chronic HDM challenge as compared to IL33 HumInmice administered HDM with an isotype control antibody during this timeperiod. The effects on frequency of eosinophils, alveolar macrophages,activated CD8 T cells, ST2+CD4+T cells and CD4/CD8 ratio in the lungobserved for the combination anti-IL33 and anti-mouse IL-4Rα antibodiesshows a trend towards greater efficacy than treatment with eitherindividual antibodies alone.

TABLE 7 Frequency of pulmonary cell infiltrate as determined by flowcytometry Mean Mean Mean Mean Mean Mean Frequency Frequency of Frequencyof Frequency of Frequency Frequency of of Alveolar Mean ActivatedActivated Activated of ST2+ Eosinophils Macrophages CD4/CD8 cells incells in cells in CD4+ cells in the live in the live T cells CD4 T cellsCD8 T cells B cells in T cells Experimental population population ratiopopulation population population population group (±SD) (±SD) (±SD)(±SD) (±SD) (±SD) (±SD) 1. 1X PBS challenge  1.45 5.05 3.00 13.12  3.260.39  3.25 (n = 5) (±0.92) (±1.64)  (±1.48)  (±9.89) (±1.64) (±1.17) (±4.15) 2. HDM challenge 17.08 2.34 6.42 49.95  9.58 4.67 32.60 11 weeks(n = 4)  (±3.94) * (±0.93)  (±2.71)  (±8.76) (±7.44)  (±1.47) **(±12.23)  3. HDM challenge 15.40 4.92 6.95 58.53 15.68 3.70 37.33 15weeks (n = 4)  (±3.99) * (±1.55)   (±0.71) ** (±5.76)  (±3.03) * (±1.44) *  (±8.98) * 4. HDM challenge 15.00 2.33 7.49 57.75 14.59 3.9037.96 15 weeks + isotype  (±3.35) * (±1.60)   (±1.28) * (±7.64) (±3.82) (±1.48) *  (±16.71) * control antibody (n = 4) 5. HDM challenge  8.517.44 4.03 48.22 13.86 1.72 19.24 15 weeks + anti- (±7.52) (±4.18) (±1.28)  (±5.66) (±5.21) (±0.72)  (±5.72) IL33 antibody (n = 5) 6. HDMchallenge 12.30 9.93 5.56 53.42 13.11 2.14 35.01 15 weeks + anti-(±7.83) (±5.18)  (±2.22)  (±6.52) (±6.26) (±1.23)   (±9.83) * mouseIL-4Rα antibody (n = 5) 7. HDM challenge  3.78 14.64  2.96 42.52  7.901.74 11.78 15 weeks + anti- (±1.60)  (±3.86) ^(†) (±0.93)  (±9.79)(±1.30) (±0.91)  (±3.73) IL33 + anti-mouse IL-4Rα antibodies (n = 5)Note: Statistical significance determined by Kruskal-Wallis One-wayANOVA with Dunn's multiple comparison post-hoc test is indicated (* = p< 0.05, ** = p < 0.01, compared to groups 1: IL33 HumIn mice, Salinechallenge; ^(†) p < 0.05, compared to group 4: IL33 Humin mice, HDMchallenge 15 weeks + Isotype control antibody).

Lung harvest for quantification of histopathology. The inflammatorypattern observed in this model is accompanied by widespread and severestructural changes in HDM-exposed lungs, with evidence of goblet cellmetaplasia, increases in sub-epithelial collagen deposition andsignificant pulmonary consolidation. These pathologies are knownfeatures of human inflammatory respiratory diseases that contribute todecline of lung function and airway hyperreactivity.

After exsanguination, the left lungs were removed and placed into platescontaining a 3 mL solution of 4% (w/v) paraformaldehyde (BostonBioproducts, # BM-155) in 1× phosphate buffered saline and stored atroom temperature for 3 days. Lung samples were then blotted dry andtransferred to tubes containing 70% ethanol for histological analysis.The samples were sent to Histoserv, Inc (Germantown, MD) for paraffinembedding, sectioning and periodic acid Schiff (PAS) or Hematoxylin andEosin (H&E) staining.

Quantification of Goblet cell metaplasia. Goblet cell metaplasia andmucus hyper-secretion are hallmarks of many pulmonary diseases includingasthma, chronic obstructive pulmonary disease, and cystic fibrosis.Excessive mucus production leads to airway obstruction and affectsseveral important outcomes such as lung function, health-related qualityof life, exacerbations, hospitalizations, and mortality in humans.PAS-positive goblet cells and total epithelial cells were counted in amillimeter length of the primary bronchus. Goblet cell metaplasia isexpressed as the frequency of PAS-positive cells in a millimeter ofbronchial epithelium (%, ±SD) as shown in Table 8.

Quantification of lung consolidation. Lung consolidation includes theaccumulation of solid or liquid material in the alveolar space. Lungconsolidation is a compound endpoint likely reflecting the combinationof cellular infiltrate, hyperplasia, and mucus production, used here asa measurement of gross pathology. The fraction of lung area occupied bythe crystal bodies was quantified on Movat pentachrome stainedparaffin-embedded lung sections using Image) software (NIH, Bethesda,MD). Using the particle analysis function, total lung area in thesection, as well as consolidated area in the section were measured. Thefraction of consolidated lung area is given by the ratio of bothmeasurements, as shown in Table 8.

Quantification of sub-epithelial fibrosis. Sub-epithelial fibrosisincludes an excess of interstitial collagen deposition beneath thepulmonary epithelium. Increased sub-epithelial fibrosis has beenreported to be specifically associated with asthma in humans. In themodel, sub-epithelial fibrosis was measured on Masson's trichromestained paraffin-embedded lung sections using HaLo software (IndicaLabs, NM). Using the Layer thickness tool, the thickness of the collagenlayer beneath the bronchial epithelium was recorded multiple times, withabout 30 μm intervals, across a millimeter of the primary bronchus.Sub-epithelial fibrosis is expressed as the mean thickness of thecollagen layer beneath the epithelium (μm, ±SD) as shown in Table 8.

Analysis of lung histopathology. As shown in table 9, there was a trendtowards an increase in goblet cell metaplasia in the lungs of IL33 HumInmice receiving HDM for 15 weeks, with or without treatment with anisotype control antibody compared to IL33 HumIn mice challenged with1×PBS alone. Similarly, there was a significant increase in lungconsolidation, as well as in sub-epithelial collagen thickness, in IL33HumIn mice receiving HDM for 15 weeks.

In contrast, there was trend towards a reduction in goblet cellmetaplasia and sub-epithelial collagen thickness, and a significantreduction in lung consolidation in IL33 HumIn mice treated with acombination of anti-IL33 and anti-mouse IL-4Rα antibodies during thelast four weeks of the chronic HDM challenge as compared to IL33 HumInmice administered HDM with an isotype control antibody during this timeperiod. The effects on goblet cell metaplasia, lung consolidation andsub-epithelial collagen thickness observed for the combination anti-IL33and anti-mouse IL-4Rα antibodies showed a trend towards greater efficacythan treatment with either individual antibodies alone.

TABLE 8 Quantification of histopathology in mouse lungs Mean sub- MeanGoblet epithelial cell metaplasia Mean lung collagen (% PAS-positiveconsolidation thickness Experimental group cells) (±SD) (% ± SD) (μm)(±SD) 1. 1X PBS challenge (n = 5) 32.94 (±43.61) 6.97 (±3.72) 25.90(±4.00) 2. HDM challenge 11 weeks 59.98 (±39.01) 70.70 (±12.94) 81.76(±25.37) * (n = 4) 3. HDM challenge 15 weeks 92.15 (±10.16) 83.21(±3.65) ** 82.12 (±23.04) * (n = 4) 4. HDM challenge 15 81.60 (±17.56)84.16 (±5.85) ** 63.11 (±11.87) weeks + isotype control antibody (n = 4)5. HDM challenge 15 weeks + 39.22 (±18.93) 58.82 (±18.26) 70.99 (±23.85)anti-IL33 antibody (n = 5) 6. HDM challenge 15 weeks + 79.82 (±25.02)57.79 (±18.72) 57.62 (±15.34) anti-mouse IL-4Rα antibody (n = 5) 7. HDMchallenge 15 weeks + 19.69 (±8.80) 35.01 (±20.68) 48.19 (±18.58)anti-IL33 + anti-mouse IL- 4Rα antibodies (n = 5) Note: Statisticalsignificance determined by Kruskal-Wallis One-way ANOVA with Dunn'smultiple comparison post-hoc test is indicated (** = p < 0.01, comparedto groups 1: IL33 HumIn mice, Saline challenge).

Serum collection for IgE and HDM-specific IgG1 levels measurement. Todetermine the total IgE concentration in the serum samples for eachmouse, a sandwich ELISA OPTEIA kit (BD Biosciences, #555248) was usedaccording to the manufacturer's instructions. Serum samples were dilutedand incubated with anti-IgE capture antibody coated on 96-well plates.Total IgE was detected by biotinylated anti-mouse IgE secondaryantibody. Purified horseradish peroxidase (HRP)-labeled mouse IgE wasused as a standard. The chromagen 3,3′,5,5′-tetramethylbenzidine (TMB)(BD OPTEIA substrate reagent set, BD, #555214) was used to detect HRPactivity. A stop solution of 1 M sulfuric acid was then added, andabsorbance at 450 nm was measured on a Molecular Devices SpectraMax M5plate reader. Data analysis was performed using Prism™ software. Themean amounts of circulating IgE levels in serum for each experimentalgroup are expressed as ng/mL (±SD) as shown in Table 9.

To determine the HDM specific IgG1 levels in the serum samples from eachmouse, an ELISA was utilized. HDM (Greer, #XPB70D3A2.5) coated plateswere incubated with serially diluted mouse serum samples, followed byincubation with a rat anti-mouse IgG1-HRP conjugated antibody (BDBiosciences, #559626). All samples were developed with a TMB solutionand analyzed as described above. Relative levels of circulating IgG1 inserum were represented as titer units (titer units were calculated bymultiplying the measured OD by a dilution factor required to achieveOD450 that was greater than two times background). The mean circulatingHDM-specific IgG1 levels in serum for each experimental group areexpressed as titer×10⁶ (±SD) as shown in Table 9.

Analysis of the circulation levels of IgE and HDM-specific IgG1. Asshown in Table 9, there was a significant increase in circulating levelsof IgE in the serum of IL33 HumIn mice receiving HDM for 15 weeks, withor without treatment with an isotype control antibody in IL33 HumIn micechallenged with 1×PBS alone. Similarly, there was a trend towards anincreased level of circulating HDM-specific IgG1 in the serum of IL33HumIn mice receiving HDM for 15 weeks. In contrast, there was asignificant decrease in circulating levels of IgE and a trend towards adecrease in circulating levels of HDM-specific IgG1 in the serum of IL33HumIn mice treated with a combination of anti-IL33 and anti-mouse IL-4Rαantibodies during the last four weeks of the chronic HDM challenge ascompared to IL33 HumIn mice administered HDM with an isotype controlantibody.

TABLE 9 Circulating levels of IgE and HDM-specific IgG1 in mouse serum.Mean circulating Mean circulating HDM-specific IgE levels IgG1 levelsExperimental group (μg/mL) (±SD) (Titer × 10⁶) (±SD) 1. 1X PBS challenge2.16 (±2.02) ND (n = 5) 2. HDM challenge 11 50.16 (±8.35) 1.18 (±0.15)weeks (n = 4) 3. HDM challenge 15 131.38 (±106.84) * 1.88 (±0.81) weeks(n = 4) 4. HDM challenge 15 193.07 (±78.96) *** 1.62 (±0.62) weeks +isotype control antibody (n = 4) 5. HDM challenge 15 45.74 (±45.74) 1.76(±0.98) weeks + anti-IL33 antibody (n = 5) 6. HDM challenge 15 11.12(±8.65) 0.99 (±0.56) weeks + anti-mouse IL-4Rα antibody (n = 5) 7. HDMchallenge 15 6.45 (±5.79) ^(†) 0.75 (±0.30) weeks + anti-IL33 +anti-mouse IL-4Rα antibodies (n = 5) Note: Statistical significancedetermined by Kruskal-Wallis One-way ANOVA with Dunn's multiplecomparison post-hoc test is indicated (* = p < 0.05, ** = p < 0.01, ***= p < 0.001, compared to groups 1: IL33 HumIn mice, Saline challenge;^(†)p < 0.05, compared to group 4: IL33 Humin mice, HDM challenge 15weeks + Isotype control antibody). ND: Not determined.

A combination of H4H9675P and anti-mIL-4Rα treatment initiated in thecontext of severe, mixed inflammation improves all inflammatoryparameters measured, reducing most to baseline levels. Additionally,additive effects are observed on some of the most pernicious endpoints,including composite lung gross pathology, goblet cell metaplasia, lungcellular infiltration, and cytokine levels. Therefore, blocking bothpathways simultaneously has the potential to impact multipleinflammatory mediators in the context of severe mixed inflammation andtissue pathology, and normalize multiple parameters to baseline.

Example 2 Genetic Variants in IL33 and its Receptor Associate with BothEosinophilic Asthma and COPD

In this Example, the relationship between previously identified asthmarisk variants at IL33 and IL1RL1 with risk of asthma, COPD, and ACOS wasexamined in the largest combined collection of such cases yet assembled,in which genetic data is linked to electronic health records. Theimportance of these variants to eosinophilic subtypes of asthma, COPD,and ACOS, as well as to related upper airway diseases such as nasalpolyps was examined. In addition, the association between predictedloss-of-function variants (pLOF) in IL1RL1 and IL33 with these diseaseswas evaluated.

Human Genetics Study Oversight. The human genetics studies wereconducted as part of the DiscovEHR study of the Regeneron GeneticsCenter (RGC) and the Geisinger Health System (GHS).

DiscovEHR Participants and Disease Definitions. At the time of thisstudy, the DiscovEHR study comprised a total of 92,323 adult individualsenrolled in the MyCode® Community Health Initiative of the GHS. For thisstudy 86,004 and 83,339 individuals of European ancestry had phenotype,and exome sequencing and genotype data, respectively for analysis.Participants were recruited from outpatient primary care and specialtyclinics. Eosinophil counts and disease diagnosis codes (theInternational Classification of Diseases, Ninth Revision [ICD-9]) wereextracted from EHRs, which covered a median of 14 years of clinicalcare. Median EHR-documented eosinophil count measurements were derivedfrom complete blood counts following removal of likely spurious valuesthat were >3 standard deviations from the intra-individual median value.Case status was assigned on the basis of ICD-9 codes if at least one ofthe following criteria were met: (1) a problem-list entry of thediagnosis code; or (2) an encounter diagnosis code entered for 2separate clinical encounters on separate calendar days. Individuals wereassigned one or more of the three case classifications (asthma, COPD andACOS) based on ICD-9 diagnosis codes.

Control patients for all binary trait analyses were defined asindividuals without a single ICD-9 diagnosis code of asthma or COPD.

Sequencing and Genotyping. Sample preparation and whole exome sequencingwere performed. In brief, exome capture was performed using eitherNimbleGen probes (Roche, SeqCap VCRome) or Integrated DNA Technologiesprobes (IDT, xGEN Exome Research panel) with additional contentaccording the respective manufacturer's recommended protocol. CapturedDNA was PCR amplified and quantified by qRT-PCR (Kapa Biosystems).Multiplexed samples were sequenced using 75 bp paired-end sequencing onIllumina v4 HiSeq 2500 or HiSeq X sequencers to a coverage depthsufficient to provide greater than 20× haploid read depth of over 85% oftargeted bases in 96% of samples (approximately 80× mean haploid readdepth of targeted bases). Raw sequence data from each Illumina HiSeq2500 run were uploaded to the DNAnexus platform for sequence readalignment and variant identification. Raw sequence data were convertedfrom BCL files to sample-specific FASTQ-files, which were aligned to thehuman reference build GRCh38 with BWA-mem. Single nucleotide variants(SNV) and insertion/deletion (indel) sequence variants were identifiedusing the Genome Analysis Toolkit. Samples with genotype rate less than10% were excluded. For final analyses, exome data was available for59,082 and 29,504 individuals of European Ancestry captured using VCRomexGEN probe sets, respectively.

Aliquots of DNA were genotyped using the Human OmniExpress ExomeBeadchip or the Global Screening Array (Illumina Corp.). For finalanalyses, Chip data was available for 56,239 and 28,500 individuals ofEuropean ancestry assayed on the Omni and GSA BeadChips respectively

Study Design and Statistical Analysis. OMNI and GSA Chip data was usedto evaluate two previously identified asthma risk variants (IL33(rs1342326) and IL1RL1 (rs1420101)) for association with obstructivelung diseases, other airway diseases, and circulating eosinophil counts.These variants were tested for association with disease under anadditive model using logistic regression in PLINK or R, including age,age², sex, smoking status, and the first four principal components ofancestry as covariates. Median EHR-documented eosinophil counts werelog-transformed and tested for association with genotypes under anadditive genetic model using linear (PLINK, R) models controlling forthe same covariates as above. All p-values correspond to additivegenetic models. Resulting summary statistics from analyses on bothplatforms were combined by meta-analysis.

Under the same statistical framework, exome data was used to identifyassociations between pLOF variants aggregated within IL1RL1 or IL33 andobstructive lung disease outcomes and eosinophil counts. At each gene,individuals were coded 0 if they did not carry any pLOF, and 1 if theywere heterozygous carriers of at least one pLOF; No homozygous pLOFcarriers for either IL1RL1 or IL33 were observed in this study.Resulting summary statistics from analyses on both platforms werecombined by meta-analysis.

A genetic risk score, reflecting the sum of risk alleles for twoindependent variants (IL33 (rs1342326) and IL1RL1 (rs1420101)), was alsoused as a predictor of obstructive lung disease outcomes and eosinophilcounts using logistic and linear regression models and the samecovariates described above. Individuals missing genotype data for eitheror both variants were excluded. The effects of carrying one, two, three,or four risk alleles were determined separately relative to individualscarrying no risk allele at either variant. Trends between increasingscore and increasing eosinophil counts or disease risk were tested usingthe linear regression and the Cochran-Armitage test, respectively.

All statistical analyses were performed with the use of PLINK software(v1.90p) or R version 3.2.1.

Confirmation of previously identified asthma risk variants in IL33 andIL1RL1 with DiscovEHR eosinophil counts and EHR-defined asthma. Clinicalcharacteristics of MyCode® participants in the DiscovEHR study aredescribed in FIG. 8 . Among 86,004 patients of European ancestry exomesequenced in this study, 13267 (15.4%) patients were diagnosed withasthma, 9783 (11.4%) patients with COPD, and 2993 (3.4%) patients withboth asthma and COPD (referred to here as asthma-COPD overlap syndrome,or ACOS). Among 83,339 patients of European ancestry with available Chipdata for this study, 12832 (15.4%) patients were diagnosed with asthma,9536 (11.4%) patients with COPD, and 2909 (3.5%) patients with ACOS.

The first large GWAS of asthma identified an intronic IL1RL1 variant(rs1420101) that was associated with both asthma and circulatingeosinophil counts, and a subsequent GWAS identified an upstream IL33variant (rs1342326) that was associated with asthma. In this study,association of rs1420101 (IL1RL1) and rs1342326 (IL33) with asthma (metaallelic odds ratio (OR_(allelic)) was confirmed (95% confidenceinterval) 1.07 (1.04-1.11), P=8.2×10⁻⁷ and Meta-OR_(allelic) 1.09(1.05-1.16), P=6.0×10⁻⁶, respectively) (FIG. 3 ).

Additionally, both variants were associated with lifetime mediancirculating eosinophil counts (n=66,776 individuals) (Meta-beta=0.0066(0.0054-0.0079) eos/ml, P=2.0×10⁻²³ and Meta-beta=0.0061 (0.0045-0.0078)eos/ml, P=2.0×10⁻¹³, respectively

IL33 and IL1RL1 associations with asthma are specific to eosinophilicsubset. Eosinophilic asthma is recognized as an important subset ofasthma, and seems to be associated with increased asthma severity andsteroid refractoriness, as well as differential responsiveness tobiologic therapies. Having confirmed the previously describedassociations between the IL33 and IL1RL1 variants and eosinophil countsas well as asthma, independently assessed as distinct phenotypes, thestudy next assessed whether these risks are connected through a specificassociation with the eosinophilic subset of asthma, and thereforeassociations in asthma patient subgroups stratified by high (>200eos/μL) and low (≤200 eos/μL) median lifetime eosinophil counts wereassessed (FIG. 3 ). Both the IL33 (rs1342326) and IL1RL1 (rs1420101)variants were significantly associated only with the eosinophilic asthmasubset (for IL33, the allelic meta odds ratio was 1.12 (1.06-1.18) inthe high eosinophil group vs. 1.04 (0.98-1.09) in the low eosinophilgroup; for IL1RL1, the allelic odds ratio was 1.07 (1.04-1.1) in thehigh eosinophil group vs. 1.02 (0.98-1.06) in the low eosinophil group)(FIG. 3 ).

Novel associations between asthma risk variants in IL33 and IL1RL1 andincreased risk of COPD and ACOS, specifically in eosinophilic subsets.In addition to the above associations with eosinophilic asthma, it wasfurther discovered that the IL33 (rs1342326) and IL1RL1 (rs1420101)variants are suggestively or marginally significantly associated withCOPD (FIG. 4 , for IL33, Meta OR_(allelic)=1.04 (0.99-1.09), P=8.9×10⁻²,and for IL1RL1, Meta-OR_(allelic)=1.04 (1-1.07), P=3.9×10⁻²) and ACOS(FIG. 5 for IL33, Meta OR_(allelic) 1.08 (1.0-1.16), P=3.8×10⁻², and forIL1RL1, 1.06 (1.0-1.12), P=4.8×10⁻²).

As with asthma, eosinophilic subsets of COPD and ACOS are associatedwith more severe disease. To determine whether the IL33 and IL1RL1associations with COPD and ACOS were also specific to eosinophilicsubtypes, as we had seen in asthma, associations between IL33(rs1342326) and IL1RL1 (rs1420101) in COPD and ACOS subgroups stratifiedby high (>200 eos/μL) and low (≤200 eos/μL) median lifetime eosinophilcount were assessed (FIGS. 4 and 5 ). Both variants were suggestivelyassociated with COPD and ACOS only in the disease subgroupscharacterized by high circulating eosinophils.

Higher burden of risk-increasing alleles in the IL33 signaling pathwayleads to larger increases in asthma, COPD and ACOS risk. Since IL33 andIL1RL1 are part of the same signaling complex, and since these variantsdisplay notable allele-dosage dependence in their risk associations whenanalyzed individually, a two-variant genetic risk score was constructedby summing the number of risk alleles at IL33 (rs1342326) and IL1RL1(rs1420101) (each individual had a score ranging from 0-4), and theassociation between the score and eosinophil counts and risk of asthma,COPD and ACOS was tested. Groups of individuals carrying each geneticrisk score were compared to the group with zero risk alleles. In testsfor trend, increasing genetic risk score was significantly associatedwith increasing eosinophil counts (FIG. 2 , P=1×10⁻³⁹) and increasingrisk of asthma (FIG. 6 , P=3.27×10⁻¹²), COPD (FIG. 6 , P=6.65×10⁻³), andACOS (P=4.3×10⁻³). The largest effects (exceeding nominal significance)were observed for patients carrying three risk alleles (FIG. 2 , foreosinophil counts, Meta beta=0.0071 (0.0057-0.0085) eos/ml, P=8.8×10⁻²⁴;for asthma (FIG. 6 ), Meta OR=1.28 (1.17-1.41), P=6.99×10⁻⁸; for COPD(FIG. 6 ), OR=1.17 (1.04-1.31), P=9.35×10⁻³); and for ACOS (FIG. 6 ),OR=1.23 (1.03-1.48), P=2.49×10⁻² (FIG. 2 ). Few individuals carried 4risk alleles, and consequently effect size estimates had wide confidenceintervals.

Associations between the two-variant score and high and low eosinophilpatient subgroups was also assessed (FIG. 7 ). In trend tests, the scorewas significantly associated with high-eosinophil subsets of asthma(P=1.37×10⁻¹⁵), COPD (P=3.9×10⁻⁸), and ACOS (P=1.8×10⁻⁵), but not withlow-eosinophil subsets of asthma, COPD, or ACOS (P>0.05 for eachdisease). The largest score-specific effects were observed for patientscarrying three risk alleles (for high eosinophil asthma, Meta-OR=1.61(1.42-1.84), P=6.33×10⁻¹³; for high eosinophil COPD, Meta-OR=1.53(1.31-1.79), P=4.76×10⁻⁸); and for high eosinophil ACOS, OR=1.7(1.34-2.14), P=1×10⁻⁵. As previously noted, there were relatively fewindividuals carrying 4 risk alleles, and respective effect sizeestimates had wide confidence intervals.

Predicted loss-of-function (pLOF) variants in IL33 are associated withdecreased circulating eosinophil counts and obstructive lung diseaserisk. In analyses of IL33 pLOF variant rs146597587, IL33 inactivation isassociated with reduced eosinophil counts (Meta-Beta=−0.02(−0.03-0.0092,P=7.3×10⁻⁵) but not significantly with a reduced risk ofeosinophilic asthma, COPD and ACOS (OR=0.82 (0.63-1.07), P=0.15, OR=0.99(0.74-1.33), P=0.94,and OR=0.93 (0.56-1.53), P=0.76, respectively) (FIG.7 ). IL1RL1 pLOF variation was not associated with risk of obstructivelung disease.

Analysis of IL1RL1 variant rs1420101 and IL33 variant rs1342326 withother airway diseases. The unified airway theory posits that asthma mayco-occur with other airway disease due to common mechanisms. Therefore,other EHR-documented diseases of the airway for association withrs1420101 and rs1342326 were tested (FIG. 9 ). The IL33 variantrs1342326 and IL1RL1 variant rs1420101 was associated with an increasedrisk of allergic rhinitis (Meta-OR 1.04 (1.01-1.08), P=0.02; Meta-OR1.04 (1.01-1.06), P=2.4×10⁻³) as well as an increased risk for nasalpolyps (Meta-OR 1.48 (1.28-1.72), P=1.2×10⁻⁷, Meta-OR 1.17 (1.04-1.33),P=1.2×10⁻²). Furthermore, the burden of these common risk variants alsosignificantly increased the risk for allergic rhinitis and nasal polys(P=1.45×10⁻⁴, P=2.48×10⁻⁷). These results are consistent with a previousreport implicating IL33 common genetic variation in nasal polyps risk.

Summary. IL33 is thought to be involved in barrier defense in epithelialtissues, including the lung epithelium, and has been implicated inasthma pathogenesis. The two variants described in this Example, IL33(rs1342326) and IL1RL1 (rs1420101), have been previously associated withasthma in several studies. These reproducible and independentassociations (IL33 is located on chromosome 9; IL1RL1 is located onchromosome 2) with both the ligand (IL33) and its specific receptor(IL1R1) suggest a role for IL33 signaling in asthma risk.

The current study significantly extends those previous findings. Byperforming whole exome sequencing and genotyping in over 83,000 adultparticipants of the DiscovEHR study associations were confirmed betweenIL33 and IL1RL1 and eosinophil counts as well as asthma, independentlyassessed as distinct phenotypes. Furthermore, a suggestive associationof the IL33 and IL1RL1 variants with increased risk of COPD and ACOS wasdemonstrated—providing a genetic link supporting possibility of a sharedmechanistic etiology between all three of these highly prevalent lungdiseases. Associations of these variants with nasal polyps and allergicrhinitis were also demonstrated. In addition, it was found that inindividuals carrying a larger burden of these risk alleles across bothloci, larger effects on disease risk were observed. Furthermore,heterozygous carriers of a rare pLOF variants in IL33 had lower medianlifetime eosinophil counts and trends reflecting about 20% decreasedrisk of asthma. It is believed that these data provide genetic evidencelinking the IL33 pathway to asthma and possibly to COPD through anallelic series that includes both risk-increasing common alleles andrisk-decreasing rare pLOF alleles.

It is believed that prior to this study, genetic variants in the IL33pathway had not been previously associated with COPD. Similarly, it isbelieved that there was no prior genetic data linking the any pathway tothe risk of the eosinophilic subsets of asthma, COPD and ACOS. Theresults of this Example suggest a link between enhanced IL33 signalingfor increased risk of the eosinophilic subtypes of asthma and COPD and,the numerically higher risk associations seen with ACOS patientssuggests that this entity at the intersection of these conditions mayindeed have special features. In addition to providing a unifyinggenetic and mechanistic link between eosinophilic subsets of heretoforedistinctly labeled obstructive lung diseases, the data also support thetenets of the “unified airway theory” that posits that eosinophilic lungdiseases may represent a continuum with related upper airway diseases.In this respect, a markedly increased risk for the IL33 variant inallergic rhinitis and nasal polyps was observed.

Although not statistically significant, the protective associations withIL33 pLOF variants described in this Example are consistent with arecent study that demonstrated that a rare loss-of-function variant inIL33 was protective in asthma, supporting the possibility thatinhibition of IL33 signaling may be an important therapeutic strategyfor obstructive lung diseases. The data, in particular, suggest a rolefor interleukin-33 blockade in the eosinophilic forms of obstructivelung diseases such as asthma and COPD, as well as for eosinophilic upperairway diseases such as allergic rhinitis and nasal polyps.

Relatedly, recent progress with biologics in the treatment of severe andsteroid-resistant asthma seems to distinguish eosinophilic disease.Multiple therapies that target interleukin-5 and interleukin-13 seem toonly benefit the eosinophilic subset of asthma patients, while anantibody (Dupilumab) that blocks both the interleukin-4 andinterleukin-13 pathways has numerically greater benefits in theeosinophilic patients, but also seems to have profound activity in thelow-eosinophil subset. These therapies also seem to have benefit innasal polyps and allergic rhinitis. Consistent with these previouslydescribed differences in the responses of eosinophilic asthma patientsto biologics therapies, the data from this Example suggests thatinterleukin-33 blockade might best target the eosinophilic subsets ofasthma, ACOS and COPD.

Although this study has certain limitations, it nonetheless represents areal-world clinical care setting, and in this population IL33 and IL1RL1genetic variation is associated with increased risk of diagnosis of bothasthma and COPD. For the purposes of personalized treatment of patients,whether one arrives at a diagnostic label of asthma, COPD, or ACOS isperhaps less important than identifying the mechanistic pathology thatis occurring in a particular patient or group of patients, and thesedata suggest that subsets of asthma, COPD and ACOS patients may in partbe driven by excess IL33 activity. Mitigating against variouslimitations of these data is the remarkable consistency of the findingsusing genetic variants in two different genes within the samepathway—parallel results were seen for variants in the gene for IL33 aswell as for its receptor. For variants in both genes, consistent riskassociations were seen across multiple related EHR-defined diseasesettings, and within these disease settings, consistent results werealso repeatedly noted specific to the eosinophilic subsets of thesediseases. Another convincing aspect of the data involves the consistentand notable allele-dependence of most of the risk associations, as wellas the added power resulting from the two-variant risk score analyses.Finally, the reciprocal findings with the IL33 pLOF variants is alsosupportive.

These data suggest that genetic variation that enhances IL33 signalingcontributes to increased risk of the eosinophilic forms of asthma, COPDand ACOS, and that pLOF genetic variants in IL33 may contribute toreduced risk of these diseases; risk of upper airway diseases such asnasal polyps also appears to be linked to IL33 signaling. Individualscarrying genetic variants that enhance IL33 signaling may represent anopportunity for precision medicine, as those particular asthma and COPDpatients may benefit most from therapeutic blockade of IL33. The dataalso raise the possibility that patients suffering from eosinophilicairway disease, regardless of subtype and variant status, may benefitfrom inhibition of IL33.

The disclosure is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims. U.S. application Ser. No. 15/827,357filed Nov. 30, 2017 is incorporated herein by reference in its entiretyfor all purposes.

1-42. (canceled)
 43. A method for treating or inhibiting eosinophilicasthma-Chronic Obstructive Pulmonary Disease (COPD) overlap syndrome(ACOS), comprising administering an IL33 antagonist or a combination ofan IL33 antagonist and an IL-4R antagonist to a subject having the IL33variant rs1342326 but not having the intronic IL1RL1 variant rs1420101,or having both the intronic IL1RL1 variant rs1420101 and the IL33variant rs1342326, wherein ACOS is treated or inhibited in the subject.44. The method according to claim 43, wherein the subject has theintronic IL1RL1 variant rs1420101.
 45. (canceled)
 46. The methodaccording to claim 43, wherein the subject has the IL33 variantrs1342326.
 47. (canceled)
 48. The method according to claim 43, whereinthe subject has the IL33 variant rs1342326, and the intronic IL1RL1variant rs1420101. 49-51. (canceled)
 52. The method according to claim43, wherein the IL33 antagonist comprises an IL33 trap.
 53. The methodaccording to claim 52, wherein the IL33 trap comprises a first IL33binding domain comprising an IL33 binding portion of IL1RL1 and a secondIL33 binding domain comprising an extracellular portion of IL-1RAcP. 54.The method according to claim 43, wherein the IL-4R antibody comprisesdupilimab. 55-60. (canceled)
 61. The method according to claim 43,wherein the ACOS is a high-eosinophil eosinophilic ACOS.
 62. The methodaccording to claim 61, wherein the subject has the intronic IL1RL1variant rs1420101.
 63. The method according to claim 61, wherein thesubject has the IL33 variant rs1342326.
 64. The method according toclaim 61, wherein the subject has the IL33 variant rs1342326, and theintronic IL1RL1 variant rs1420101.
 65. (canceled)
 66. The methodaccording to claim 61, wherein the IL33 antagonist comprises an IL33trap.
 67. The method according to claim 66, wherein the IL33 trapcomprises a first IL33 binding domain comprising an IL33 binding portionof IL1RL1 and a second IL33 binding domain comprising an extracellularportion of IL-1RAcP.
 68. The method according to claim 61, wherein theIL33 antagonist comprises an antibody or antigen-binding fragmentthereof that specifically binds to IL33.
 69. The method according toclaim 68, wherein the antibody, or antigen-binding fragment thereofthereof, that specifically binds to IL33 comprises the complementaritydetermining regions of a heavy chain comprising the amino acid sequenceof SEQ ID NO: 274 and the complementarity determining regions of a lightchain comprising the amino acid sequence of SEQ ID NO:
 282. 70. Themethod according to claim 61, wherein the IL-4R antagonist comprises anantibody or antigen-binding fragment thereof that specifically binds toIL-4R.
 71. The method according to claim 70, wherein the antibody, orantigen-binding fragment thereof, that specifically binds to IL-4Rcomprises the complementarity determining regions of a heavy chaincomprising the amino acid sequence of SEQ ID NO: 337 and thecomplementarity determining regions of a light chain comprising theamino acid sequence of SEQ ID NO:
 338. 72. The method according to claim71, wherein the IL-4R antibody comprises dupilimab.